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Woven silk fabric-reinforced silk nanofibrous scaffolds for regenerating load-bearing soft tissues
Acta Biomaterialia 10 (2014) 921–930
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Woven silk fabric-reinforced silk nanofibrous scaffolds for regenerating load-bearing soft tissues F. Han a,b, S. Liu a,1, X. Liu a,1, Y. Pei a, S. Bai a, H. Zhao a, Q. Lu a,⇑, F. Ma b,⇑, D.L. Kaplan c, H. Zhu d a
National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China Key Lab of Rubber–Plastics (QUST), Ministry of Education, College of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042, China c Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA d Research Center of Materials Science, Beijing Institute of Technology, Beijing 100081, China b
a r t i c l e
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Article history: Received 19 April 2013 Received in revised form 3 September 2013 Accepted 23 September 2013 Available online 1 October 2013 Keywords: Silk Dermal substitute Mechanical properties Scaffolds Suture retention
a b s t r a c t Although three-dimensional (3-D) porous regenerated silk scaffolds with outstanding biocompatibility, biodegradability and low inflammatory reactions have promising application in different tissue regeneration, the mechanical properties of regenerated scaffolds, especially suture retention strength, must be further improved to satisfy the requirements of clinical applications. This study presents woven silk fabric-reinforced silk nanofibrous scaffolds aimed at dermal tissue engineering. To improve the mechanical properties, silk scaffolds prepared by lyophilization were reinforced with degummed woven silk fabrics. The ultimate tensile strength, elongation at break and suture retention strength of the scaffolds were significantly improved, providing suitable mechanical properties strong enough for clinical applications. The stiffness and degradation behaviors were then further regulated by different after-treatment processes, making the scaffolds more suitable for dermal tissue regeneration. The in vitro cell culture results indicated that these scaffolds maintained their excellent biocompatibility after being reinforced with woven silk fabrics. Without sacrifice of porous structure and biocompatibility, the fabric-reinforced scaffolds with better mechanical properties could facilitate future clinical applications of silk as matrices in skin repair. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Rapid wound closure following large-surface and deep injuries protects wounds from infection and dehydration and prompts skin regeneration. By far, autologous skin graft is still the ideal approach to close or replace irreparably damaged skin. However, a lack of donor skin grafts has led to a search for alternative substitutes to replace damaged tissue [1,2]. A range of dermal substitutes have been developed for skin regeneration, including decellularized dermis from human or animals, scaffolds composed of natural and/or artificial polymers such as collagen, fibronectin, polycaprolactone and poly(lactic-co-glycolic acid) [2,3]. Besides interconnecting porous structure, good biocompatibility and biodegradation to provide suitable space and environments for cellular and tissue growth, a qualified scaffold should also provide the desired mechanical strength to bear suture tension in the implantation process and maintain the structure and functional properties of the tissue after implantation [4,5]. A strong correlation between scaffold stiffness ⇑ Corresponding authors. Tel.: +86 512 67061649 (Q. Lu), +86 532 84022950 (F. Ma). E-mail addresses: [email protected] (Q. Lu), [email protected] (F. Ma). 1 The authors contributed equally with the first author.
and cell behavior was revealed recently, which showed that scaffold stiffness influences the differentiation of mesenchymal stem cells [6–8]. These findings imply more rigorous mechanical requirements for the scaffolds used in load-bearing soft tissues that have low stiffness with high strength to undergo loading and strength in tension. However, obtaining the appropriate stiffness as well as strength in scaffolds is still a challenge in general for single natural or synthetic polymer. As a natural biomaterial, silk has been used as biomedical suture for decades. Recently, silk porous biomaterials have been applied in soft tissue regeneration, including skin, nerve, blood vessel, ligament and cartilage repair, owing to its impressive biocompatibility, biodegradation, low inflammatory reactions and high processability [9–16]. A number of methods, such as freeze-drying, salt leaching, gas forming and electrospinning, have been developed to prepare regenerated silk scaffolds with various conformations and microstructures [17–21]. All these porous silk biomaterials are biocompatible and provide favorable microenvironments for both cellular infiltration and growth in vitro and tissue regeneration in vivo. However, similar to other natural biomaterials, the regenerated silk scaffolds exhibit inferior mechanical properties, particularly for applications requiring strength in tension. Degummed silk fibers, that is, sericin-extracted
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.09.026
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fibers, have been investigated particularly for the purposes of ligament and tendon repair, because of their extraordinary strength. But cellular attachment and growth as well as ease of implantation may be limited for a fiber-only material [22–24]. Conversely, the stiffness of the degummed fibers (tensile strength 24.88 ± 4.95 MPa [25]) is also too high for some soft tissues such as skin (in vivo elastic modulus 0.13–0.66 MPa [26]), which might result in a negative influence on tissue regeneration. The special microstructure of soft tissues offers a possible template for designing scaffolds with suitable stiffness and strength. Many soft tissues can be viewed as hydrogel matrixes containing cells and reinforcing fibers [27]. Therefore, a technique that has developed significantly in the field of scaffold fabrication for tissue regeneration is the use of fiber reinforcement within cell-seeded natural biomaterial scaffold systems [4,28]. Recently, silk and collagen sponges were incorporated in knitted silk fiber scaffold to improve cell proliferation and anterior cruciate ligament reconstruction [9,29]. However, this study designed a silk fiber-reinforcing scaffold system composed mainly of silk fibers rather than porous scaffold, which might be problematic in applications such as nerve (in situ stress 0.05 MPa [30]) and dermis because of high stiffness and lack of porous structures in the system. In order to satisfy the requirements of regenerating load-bearing soft tissues, it is necessary to design a new silk fiber-reinforcing system composed mainly of porous scaffolds without the sacrifice of enough strength from fibroin fibers. Nanofiber scaffolds have advanced as tissue engineering scaffolds, because they mimic the fibrous nanostructure of native extracellular matrix (ECM) [31–33,21]. In a recent study, freezedried nanofiber silk scaffolds were prepared directly by regulating the self-assembly of fibroin in aqueous solution [34]. Based on this development, it is possible to achieve an excellent microenvironment with fibroin-based scaffolds for different soft tissue regeneration, if the strength of the scaffolds is further improved. Therefore, the long-term goal of this work is to fabricate a mechanically viable, silk-based scaffold system that is suitable for different soft tissue regeneration. In an attempt to improve the mechanical properties, the present authors investigated the effectiveness of reinforcing silk scaffold with silk woven fibers. Unlike the previous study, the fiber-reinforcing system is composed mainly of porous scaffold rather than fibers. The stiffness of the system is from fibroin porous scaffold and could be regulated by various after-treatments such as water annealing and methanol immersion. The cell compatibility was also investigated to confirm the influence of silk fibers on the biocompatibility of the fiber-reinforcing scaffold system, indicating that this scaffold is suitable for producing a dermal substitute for skin regeneration.
2. Experimental
the hood for 72 h at room temperature to concentrate the solution with controlled rate. During the concentrating process, silk fibroin self-assembled to nanofibers that would facilitate porous structure formation in following lyophilization. Then the 25–30 wt.% concentrated silk solution was obtained and finally diluted to 2–3 wt.% with deionized water. 2.2. Preparation of fiber-reinforced scaffolds Raw Bombyx mori silk fibers were used in this study. The woven fabrics were produced by the multiple-warp weaving method on a weaving machine (Longda, ASL2300, Tianjin, China) to form a plain weaving structure. Then the fabrics were boiled for 20 min in an aqueous solution of 0.02 M Na2CO3 to remove the sericin proteins. The sericin-free woven silk fabrics were placed on the bottom of a polystyrene mold and then immersed in diluted silk solution obtained from the procedure in Section 2.1. The mold filled with the silk solution was directly placed at 20 °C for 12 h and lyophilized for 48 h. After lyophilization, the final step was to induce crystallinity. Crystallinity can be induced by water annealing or methanol immersion to achieve water stability, according to published procedures [34]. Water annealing is the process in which the silk scaffolds are incubated in a humid environment for several hours to facilitate the transformation from random structure to silk I and silk II crystals. Briefly, the scaffolds were placed on a removable platform under which water was filled in a desiccator with a 25 in. Hg vacuum for 6 h. Unlike the water annealing process, the methanol annealing process could achieve more silk II formation by immersing the samples in 80% (v/v) methanol for 30 min. The changes in crystal structures in the treated scaffolds further resulted in the adjustment of mechanical properties of the scaffolds. The water-annealed and methanol-annealed fiber-reinforced scaffolds are termed WA-C and MA-C, respectively. In these fiber-reinforced systems, the weight content of woven silk fabrics was 30.6 ± 4.3%, indicating that the systems were composed mainly of silk porous scaffolds. As controls, woven fabrics and silk scaffolds without fabrics were also prepared under the same process and treated by water annealing or methanol immersion, which are termed WA-F, MA-F, WA-S, MA-S. The procedure of preparation of fiber-reinforced scaffolds is shown in Supporting Fig. S1. 2.3. Fourier transform infrared spectroscopy The structures of the silk woven fabrics and silk sponge in the combined scaffolds were analyzed by Fourier transform infrared (FTIR) spectroscopy on a NICOLET FTIR 5700 spectrometer (Thermo Scientific, FL, America) equipped with a MIRacle™ attenuated total reflection Ge crystal cell in reflection mode. Slices 1 mm thick were cut from the scaffolds with a razor blade in liquid nitrogen. For each measurement, 64 scans were coded with resolution 4 cm1, with the wave number ranging from 400 to 4000 cm1.
2.1. Preparation of silk solutions 2.4. X-ray diffraction Bombyx mori silk solutions were prepared according to a recently published procedure [35]. Cocoons were boiled for 20 min in an aqueous solution of 0.02 M Na2CO3, and then rinsed thoroughly with deionized water to extract sericin proteins. The degummed silk was dissolved in 9.3 M LiBr solution at 60 °C for 4 h, yielding a 20 wt.% solution. This solution was dialyzed against deionized water using Slide-a-Lyzer dialysis cassettes (Pierce, molecular weight cut-off 3500 D) for 72 h to remove the salt. Then the solution was centrifuged at 9000 rpm for 20 min at 4 °C to remove silk aggregations formed during the process. The final concentration of aqueous silk solution was 7 wt.%, determined by weighing the remaining solid after drying at 60 °C. This fresh silk solution was put into the oven for 24 h at 60 °C and then put into
X-ray diffraction (XRD) was also performed on samples with an X-ray diffractometer (X0 Pert-Pro MPD, PANalytical, Almelo, Holland) with Cu Ka radiation at 40 kV, 30 mA with a scanning rate of 0.6° min1. Before examination, the dried samples were pressed into sheets with a forcing compressor. 2.5. Scanning electron microscopy The surface and cross-section images of the fabrics and silkbased scaffolds were examined by scanning electron microscopy (SEM) (Model S-4800, Hitachi, Tokyo, Japan) at 3 kV, to avoid the destruction of silk structure. Before SEM examination, the dried
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scaffolds were cut with a razor blade in liquid nitrogen and then sprayed with platinum. 2.6. Mechanical testing The suture retention strengths in dry and wet states were obtained according to previously reported methods [36]. One end of a scaffold sheet sample was clamped (length 10 mm, width 5 mm, thickness 1.5 mm) and a 5–0 polyester suture (Jinhuan Company, Shanghai, China) was passed through the scaffold sheet, 2 mm from the edge. The suture was pulled at a rate of 50 mm min1 until pull out. This test was repeated five times per scaffold at different sites. Tensile mechanical testing was performed on the scaffolds in the dry and wet states using a load testing machine (Model 3366, Instron, Norwood, MA). Samples (length 50 mm, width 10 mm, thickness 1.5 mm) for tensile tests were clamped with a 30 mm inter-clamp distance and pulled at a rate of 10 mm min1 until rupture. Maximum stress and strain at rupture were measured. The Young’s modulus representing the elasticity was obtained by measuring the slope of the stress–strain curve in the elastic region. For measurement in the wet state, the samples were soaked in PBS at 37 °C for 1 h and then measured. Since the silk scaffolds could be fully saturated when immersed in water for >10 min, the samples in the present study were soaked in water for 1 h to ensure the achievement of saturated states. The analysis was repeated five times for each sample. Then the representative images were presented. 2.7. Silk degradation in vitro Water-insoluble scaffolds were incubated at 37 °C in 40 ml PBS containing 5 U ml1 protease XIV. Each solution contained an
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approximately equivalent mass (40 ± 5 mg) of silk scaffold with 30.6 ± 4.3% woven silk fabrics. At designated time points, samples were rinsed with deionized water and dried by lyophilization and prepared for mass balance assessment. The analysis was repeated five times for each sample. The morphologies after degradation were also examined using a Hitachi S-4800 SEM (Model S-4800, Hitachi, Tokyo, Japan).
2.8. Cell compatibility Homo sapiens dermal fibroblasts Hs 865.Sk cells obtained from ATCC (American Type Culture Collection) were used to assess the cytocompatibility of the scaffolds. Cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS), 1% IU ml1 streptomycin–penicillin (all from Invitrogen, Carlsbad, CA). Amniotic fluid-derived stem cells, with a capacity to differentiate into multiple cells and avoid ethical concerns associated with embryonic stem cells, were studied to assess the cytocompatibility of the scaffolds. Samples of amniotic fluid were obtained from the First Affiliated Hospital of Soochew University (Suzhou, Jiangsu, China) following routine amniocentesis carried out on pregnant women at 15–35 weeks of gestation. All the procedures were performed following the guidelines established by the First Affiliated Hospital of Soochew University and the First Affiliated Hospital of Soochew University Ethics Committee; written consent to use the amniotic fluid for research purposes was obtained from each woman. The isolation of human amniotic fluid-derived stem cells has been described previously [37]. Amniotic fluid-derived stem cells were cultured in a-Modified Eagle Medium supplemented with 10% FBS, 1% IU ml1 streptomycin– penicillin (all from Invitrogen, Carlsbad, CA) and growth factor: Basal, C Frozen Supplement (Irvine Scientific, Santa Ana, CA). The
Fig. 1. Microstructures of silk scaffolds and woven silk fabric-reinforced scaffolds: (a) SEM image of silk scaffolds; (b) SEM image of degummed woven fabrics; (c) microstructure of silk scaffolds near woven fabrics (arrows indicate the woven fabrics); and (d) microstructure of silk scaffolds away from woven fabrics.
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medium was replaced every 3 days, and the cultures were maintained in a humidified incubator at 37 °C and 5% CO2. Different methods, including steam, ethanol and gamma radiation, have been used to sterilize silk-based biomaterials [38]. However, considering that random and silk I structures of the water-annealed scaffolds could transform into silk II structure in steam or ethanol sterilization processes, gamma radiation was chose to sterilize the samples. The scaffolds (thickness 2 mm) were punched into small disks with diameter 8 mm and then sterilized by c radiation at room temperature, with 25 kGy from a 60Co Gammacell 220 facility, which had a dose rate of 2 kGy h1. After pre-conditioning with the culture medium overnight, the samples were transferred into a non-tissue culture treated 96-well plate and seeded with Hs 865.Sk cells at a density of 1.6 105 well1. Three hours after cell seeding, samples were transferred into a non-tissue culture-treated 6-well plates, and 5 ml complete culture medium was added to each well for up to the indicated time points. 2.9. DNA content To study cell proliferation on the scaffolds, samples were harvested at the indicated time point (from day 1 to day 12), and digested with proteinase K buffer solution for 16 h at 56 °C, as described previously [39]. The DNA content was determined using the Quant-iTTM PicoGreen dsDNA assay, following the protocols of the manufacturer (Invitrogen, Carlsbad, CA). Samples (n = 5) were measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm, using a BioTec Synergy 4 spectrofluorometer (BioTec, Winooski, UK). The amount of DNA was calculated by interpolation from a standard curve prepared with lambda DNA in 10 10–3 M Tris–HCl (pH 7.4), 5 10–3 M NaCl, 0.1 10–3 M EDTA over a range of concentrations.
3. Result and discussion 3.1. Morphology of fabric-reinforced silk scaffolds The morphology changes of silk in the treated solution are shown in Fig. S2. Through a slowly concentrating process, the nanoparticles were transformed into nanofibers in silk solution (Fig. S2A and B), which restrained the formation of lamellar structures [34] and then achieved excellent porous structures after lyophilization (Fig. 1A). The ECM provides a framework for scaffold design in which nanoscale and microscale dimensions of the physical structures are important, including nanofibrous architectures as well as specific porous structures [31–33,21]. Previous studies have confirmed that silk nanofiber formation could further improve the biocompatibility of silk scaffolds, which would provide better microenvironments for soft tissue regeneration. However, the fragile mechanical properties of the regenerated silk scaffolds restrained its clinical applications in tissue regeneration [17–21]. Therefore, the woven fabrics (Fig. 1B) were introduced into the scaffolds to afford enough mechanical strength for clinical applications in soft tissue regeneration (Fig. 1C). The degummed woven fabrics had a smooth surface after the extraction of sericin (Fig. 1B). Compared with silk fibroin scaffolds, the fabric-reinforced scaffolds had a similar interconnected porous structure, with pore
2.10. Cell viability and morphology The morphology of Hs 865.Sk cells seeded on the scaffolds was examined by confocal microscopy. Briefly, the cell-seeded scaffolds were washed three times with PBS (pH 7.4) and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 30 min, followed by further washing. The cells were permeabilized using 0.1% Triton X-100 for 5 min and incubated with FITC-phalloidin (SigmaAldrich, St. Louis, MO) for 20 min at room temperature, followed by PBS washing and finally staining with DAPI (Sigma-Aldrich, St. Louis, Mo) for 1 min. Representative fluorescence images (10) of stained samples were obtained by confocal laser scanning microscopy (CLSM, Olympus FV10 inverted microscope, Nagano, Japan) with excitation/emission at 358/461 nm and 494/518 nm. The images of the scaffolds were captured from surface deep to 100 lm at increments of 10 lm, respectively. The cell morphology on the scaffolds was confirmed by SEM. After harvest, the cellseeded scaffolds were washed three times with PBS and fixed in 4% paraformaldehyde at room temperature, and then the scaffolds were washed three times with PBS again. Fixed samples were dehydrated through a gradient of alcohol (50%, 70%, 80%, 90%, 100%, 100%) followed by lyophilization. Specimens were examined using a Hitachi Model S-4800 SEM (Hitachi, Tokyo, Japan). 2.11. Statistical methods All statistical analyses were performed using SPSS v.16.0 software. Comparison of the mean values of the data sets was performed using two-way ANOVA. Measures are presented as means ± standard deviation, unless otherwise specified, and P < 0.05 was considered significant.
Fig. 2. (A) FTIR spectra and (B) XRD data for silk fabrics and scaffolds after different treatments: (a) degummed silk fabrics; (b) water-annealed silk scaffolds; (c) waterannealed fabric-reinforced scaffolds; (d) methanol-annealed silk scaffolds; and (e) methanol-annealed fabric-reinforced scaffolds.
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size ranging from 200 to 300 lm, indicating that the addition of silk woven fabrics did not have a negative effect on porous structure formation (Fig. 1C and D). The scaffolds were then treated by water or methanol annealing to achieve water stability without significant porous structural changes (data not shown). Importantly, the weight ratio of the fabrics was significantly decreased compared with that of previous silk fiber-reinforced sponges used in ligament reconstruction [40], making it possible to be used in soft tissue regeneration. Although the weight ratio of fabrics was 30% in the scaffolds, the fabrics were only located near the surface of the scaffolds. The woven fabric was a thin lamellar, meaning
Fig. 3. Suture retention strengths of silk scaffolds: WA-S, water-annealed silk scaffolds; MA-S, methanol-annealed silk scaffolds; WA-C, water-annealed fabricreinforced scaffolds; and MA-C, methanol-annealed fabric-reinforced scaffolds (⁄P<0.05).
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that most part of the scaffold was porous sponge. The special structure might reduce the influence of stiff fabrics on cell behaviors. 3.2. Structural analysis Two major structural models, termed silk I and silk II, exist in silk fibroin. Alpha-helix dominated the silk I structure existing within the silkworm gland just before spinning. Silk II is the insoluble anti-parallel beta-sheet crystal conformation, which forms after the spinning of silk fibers from the spinneret of the silkworm [41]. The physical properties of silk fibers, such as mechanical, thermal, optical and dielectric properties, vary, depending on the structure of the silk and on the content of beta-sheet crystals. Several methods are used to induce beta sheet crystallization in silk biomaterials, such as methanol annealing and steam-autoclaving [41]. The structures of the scaffolds were determined by FTIR (Fig. 2A). The IR spectral region within 1700–1500 cm1 is assigned to absorption by the peptide chains of amide I (1700–1600 cm1) and amide II (1600–1500 cm1), which have usually been used for the analysis of secondary structures of silk fibroin. The peaks at 1610–1630 cm1 (amide I) and 1510–1520 cm1 (amide II) are characteristic of silk II secondary structure, while the absorptions at 1648–1654 cm1 (amide I) and 1535–1542 cm1 (amide II) are indicative of silk I conformation [35,42]. Similarly to many previous studies [23,24,28,29], the silk fabrics are mainly composed of stable silk II structure without conformational changes after water annealing or methanol annealing treatments. In order to avoid the overlap between silk fabrics and porous sponges, the sponges were torn from the scaffolds and measured independently before and after the treatments. The spectra of WA-S and the silk sponge part of WA-C showed strong peaks at 1650 cm1 and 1535 cm1, which represented a typical silk I structure. After treatment with 80% methanol for 30 min, the strength of the peak at 1625 cm1 significantly increased, implying silk II formation. These results
Fig. 4. Stress–strain curves of samples in the dry and wet states: (A) water-annealed samples in dry state; (B) water-annealed samples in wet state; (C) methanol-annealed samples in dry state; (D) methanol-annealed samples in wet state; (a) silk scaffolds; (b) fabric-reinforced silk scaffolds and (c) woven fabrics alone.
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3.3. Mechanical properties
Table 1 Mechanical properties of silk scaffolds in dry and wet states. Scaffolds
Tensile strength (MPa)
Strain at break (%)
Modulus (MPa)
Dry-WA-F Dry-MA-F Dry-WA-S Dry-MA-S Dry-WA-C Dry-MA-C Wet-WA-F Wet-MA-F Wet-WA-S Wet-MA-S Wet-WA-C Wet-MA-C
2.29 ± 0.04 2.39 ± 0.02 0.11 ± 0.03 0.13 ± 0.04 2.97 ± 0.57 3.39 ± 0.63 1.70 ± 0.01 1.74 ± 0.08 0.04 ± 0.003 0.05 ± 0.01 2.01 ± 0.16 2.11 ± 0.11
15.64 ± 1.23 16.09 ± 1.36 6.33 ± 1.48 6.73 ± 2.22 24.0 ± 2.41 29.0 ± 1.57 20.92 ± 1.64 20.89 ± 1.33 20.87 ± 1.94 20.27 ± 2.05 35.63 ± 0.68 41.57 ± 2.50
1.86 ± 0.19 2.51 ± 0.23 0.66 ± 0.20 0.77 ± 0.17 4.05 ± 0.48 4.40 ± 0.15 0.37 ± 0.08 0.62 ± 0.10 0.22 ± 0.06 0.53 ± 0.03 0.34 ± 0.01 0.60 ± 0.05
indicated that the silk fabrics had no significant inhibition on conformational transition in different treatment processes. The structural changes were confirmed by XRD results (Fig. 2B). Silk fabrics showed silk II crystal structure, with a sharp peak at 20.2° (II). The WA-S and sponge part of WA-C were characterized by diffraction peaks at 2h values of 12.2° (I), 19.7° (I), 24.7° (I) and 28.2° (I), indicating silk I structure [43]. After methanol annealing, the silk I peaks at 12.2°, 19.7°, 28.2° disappeared, and the strength of the silk II peak at 20.2° increased, indicating silk II formation in MA-S and the sponge part of MA-C. The structural study implied that the secondary structures could be regulated by different treatments, which resulted in the changes in mechanical properties and degradation behaviors of the fabric-reinforced scaffolds.
Suture retention is a critical factor in the design of scaffolds used in different tissue regeneration, as it directly relates to the success of the implant. Various tissues, experiencing different load ranges, have different requirements for suture retention strength. The suture retention strength of regenerated silk fibroin scaffolds is usually inferior to that of requirements in clinical applications, which even results in the failure of an implant. In this study, woven silk fabrics were used to withstand suturing forces. The suture retention strengths of the WA-C scaffolds and MA-C scaffolds were 3.7 ± 0.6 N and 3.7 ± 0.3 N in the dry state, and then slightly decreased to 3.2 ± 0.5 N and 3.1 ± 0.8 N in the wet state. The retention strengths showed no significant difference between water annealing and methanol annealing treatments. But the fabric-reinforced scaffolds showed better retention strength than the scaffolds without fabric (<0.6 N in dry and wet states), confirming the critical function of woven fabrics for withstanding tensile forces during suturing (Fig 3). Although the suture retention strength of fabricreinforced scaffolds is still significantly inferior to reticular dermis (46.2 N) and the decellularized dermis ECM (47.0 N) [44], considering that the generally accepted adequate suture retention strength for small blood vessel in surgery is >1.8 N [36], the present authors’ fabric-reinforced scaffolds might be acceptable for clinical dermis implants. Further study is necessary to improve the suture retention strength of silk-based scaffolds through regulating the conformations and nanostructures of silk. Besides suture retention strength, the tensile properties of silk scaffolds also improved after the addition of silk woven fabrics
Fig. 5. (A) Degradation behaviors of silk scaffolds in 5 U ml1 protease XIV solution. (a) WA-S, water annealed silk scaffolds; (b) WA-C, water annealed fabric-reinforced silk scaffolds; (c) MA-S, methanol annealed silk scaffolds; (d) MA-C, methanol annealed fabric-reinforced silk scaffolds; (e) WA-F, water annealed woven fabrics; (f) MA-F, methanol annealed woven fabrics. (B) SEM images of fabric-reinforced silk scaffolds incubated in protease XIV solution at 37 °C for 2 h: (a) WA-C; (b) MA-C.
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(Fig. 4 and Table 1). The ultimate tensile strengths (UTS) of WA-C and MA-C scaffolds were 3–4 MPa, above twenty times the strength of the corresponding scaffolds (WA-S, 110 KPa; MA-S, 130 KPa) in the dry state. The tensile strength of the hydrated fabric-reinforced scaffolds decreased slightly to 2.0–2.1 MPa, which was still significantly higher than that of scaffolds without woven fabric in the wet state (WA-S, 40 KPa; MA-S, 50 KPa). The higher elongations at break were also achieved for WA-C and MA-C scaffolds, which were >20% in the dry state and then further increased to >35% in the wet state. In addition, the mechanical properties of the woven silk without porous sponges were also investigated. The tensile strengths and elongations at break were 2.29–2.39 MPa and 15.64–16.09% in the dry state and then changed to 1.70–1.74 MPa and 20.89–20.92% in the wet state (Table 1), which was also slightly inferior to the fabric-reinforced scaffolds. The
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results further confirmed that better mechanical properties could be achieved through the fabric-reinforcing method. Scaffold stiffness is an important mediator of cell behavior to regulate cell signaling and, eventually, the formation and maintenance of tissues [45]. Many soft tissues are composite with collagen fibrils reinforcing hydrogel to form soft but strong matrix. In the present fabric-reinforcing scaffolds, woven fabrics afforded remarkably high strength, while silk sponge maintained low stiffness to simulate the microenvironment in vivo. Table 1 shows that the modulus of fabric-reinforced scaffolds in the wet state was similar to the corresponding scaffolds without fabrics and could be further regulated by different after-treatments. For example, the modulus of WA-C was 0.34 MPa, which changed to 0.60 MPa for MA-C in the wet state. The results indicated that the stiffness of the fabric-reinforcing scaffolds could be modulated with various
Fig. 6. Confocal microscopy of amniotic fluid-derived stem cells cultured on different scaffolds at day 1 and day 12: (A1) water-annealed silk scaffolds (WA-S), day 1; (A2, A3) water-annealed silk scaffolds (WA-S), day 12; (B1) water-annealed fabric-reinforced silk scaffolds (WA-C), day 1; (B2, B3) water-annealed fabric-reinforced silk scaffolds (WA-C), day 12; (C1) methanol-annealed silk scaffolds (MA-S), day 1; (C2, C3) methanol-annealed silk scaffolds (MA-S), day 12; (D1) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 1; (D2, D3) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 12. The blue color (DAPI) represents silk scaffolds and cell nucleus, while the green color (FITC labeled phalloidin) represents actin cytoskeleton.
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after-treatments to satisfy the specific requirements of different tissue regenerations without sacrifice of mechanical strength. The mechanical properties of skin in vivo and in vitro have been investigated by different groups. Although the results are generally different, owing to the changes of specimen location and test methods, recent study showed that the mean tensile strength, failure strain and elastic modulus of in vitro human skin from the back were 21.6 MPa, 54% and 83.3 MPa, respectively [46]. These mechanical properties were significantly superior to those of the present fabric-reinforcing scaffolds, implying that the scaffolds cannot replace the real dermis. However, the mechanical properties of skin in vitro were measured in the high-strain range, while the real skin maintained its function in the low-strain range. Therefore, it is more feasible to compare the mechanical properties of the fabric-reinforcing scaffolds and the skin in vivo. Interestingly, the moduli of WA-C and MA-C were 0.34 MPa and 0.60 MPa in the wet state, which were similar to the modulus of the skin in vivo tension test (0.13–0.66 MPa) [26]. Considering the influence of scaffold stiffness on regulating cell behavior and tissue regeneration, the present scaffolds may provide a suitable microenvironment for skin regeneration.
observed on the scaffolds. After day 12, cells increased and formed a continuous monolayer on the surface of the porous walls. Confocal microscopy and DNA content confirmed the cytocompatibility of the scaffolds (Figs. 7 and 8). Cells proliferated significantly and interacted to form aggregates at day 12. The cytocompatibility of the fabric-reinforced scaffolds showed no significant difference compared with that of scaffolds without fabrics, confirming that reinforcing silk scaffolds with silk fabric is a feasible way to improve the mechanical properties of silk-based scaffolds without sacrifice of cytocompatibility. Moreover, the cell compatibility assay was repeated using human dermal fibroblasts Hs 865.Sk cells and the same results were found (Figs. S4–S6). Considering the possible application of the scaffolds in skin regeneration, human dermal fibroblast cells were also cultured on the scaffolds, a common and valid way to assess the biocompatibility of biomaterials in skin repair, to assess the feasibility of the scaffolds. Similar to stem cells, human dermal fibroblasts Hs 865.Sk cells also showed
3.4. Degradation After incubation in protease XIV solution (5 U ml1) at 37 °C for 24 h, WA-S scaffold with a low content of b-sheet was almost completely degraded, while only silk fabrics remained in the WA-C scaffold, implying inconsiderable influence of the fabrics on the degradation behavior of the sponge part. Following the increase in b-sheet content after methanol treatment, the weight losses were 52% for MA-S and 39% for MA-C (Fig. 5A). The results indicated that the degradation behaviors of the scaffolds could be regulated through controlling crystal structure by different aftertreatments. Considering little degradation of silk fabrics and the content of fabrics in MA-C scaffolds (30 wt.%), the degradation ratio of the porous sponge part was 50% in MA-C, similar to that of MA-S, which confirmed the minimal effect of the fabrics. The morphology changes of the scaffolds in the degradation process were also investigated (Fig. 5B). The nanofibrous structure appeared on the surface of the macropore walls following degradation, which might promote cell adhesion, growth and migration in vitro or in vivo [34]. The mechanical properties of the fabric-reinforced scaffolds and the woven fabric in the degradation process were also measured. After the scaffold part had almost degraded, the mechanical properties of the fabric-reinforced scaffolds slightly decreased and were similar to those of the woven fabric after the same degradation treatment. The results supported the theory that the woven fabric endured the main mechanical tension in the fabric-reinforced scaffolds. 3.5. Cell compatibility Dermal tissue engineered constructs can be achieved based on these scaffolds. The present authors’ further work involves inducing targeted differentiation of stem cells on these scaffolds. Amniotic fluid stem cells show multilineage differentiation potential into various tissue types such as skin, cartilage, cardiac tissue, nerves, muscle and bone. Moreover, compared with other stem cell sources, amniotic fluid-derived stem cells possess immunosuppressive activity [47]. Therefore, in the present study, amniotic fluid stem cells were chosen to analyze the biocompatibility of the scaffolds. Amniotic fluid-derived stem cells were cultured in the scaffolds with and without silk fabrics, respectively. All the scaffolds showed excellent cytocompatibility (Fig. 6). The amniotic fluid-derived stem cells adhered and grew well on the surfaces of the scaffolds from day 1 to day 12. At day 1, only a few cells were
Fig. 7. SEM images of Hs 865.Sk cells cultivated on different scaffolds at day 1 and day 12: (A) water-annealed silk scaffolds (WA-S), day 1; (B) water-annealed silk scaffolds (WA-S), day 12; (C) water-annealed fabric-reinforced silk scaffolds (WA-C), day 1; (D) water-annealed fabric-reinforced silk scaffolds (WA-C), day 12; (E) methanolannealed silk scaffolds (MA-S), day 1; (F) methanol-annealed silk scaffolds (MA-S), day 12; (G) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 1; (H) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 12.
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Appendix B. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1 and 6, are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi: http://dx.doi.org/10.1016/ j.actbio.2013.09.026.
References
Fig. 8. Amniotic fluid-derived stem cells proliferation on silk scaffolds: WA-S, water annealed silk scaffolds; WA-C, water annealed fabric-reinforced silk scaffolds; MAS, methanol annealed silk scaffolds; and MA-C, methanol annealed fabric-reinforced silk scaffolds. Error bars represent mean ± standard deviation with n = 5.
excellent growth and proliferation behaviors on the scaffolds (Figs. S4–S6). Although it is important to study further the differentiation behavior of the amniotic cells into skin cell types on the fabric-reinforced scaffolds, the present results have proved that the scaffolds have good biocompatibility for skin regeneration, which is critical for future clinical applications. Further study in the near future will be focused on the directed differentiation of amniotic fluid stem cells into skin cell types by adding certain factors to provide better microenvironments for skin regeneration.
4. Conclusions Degummed silk fabric was used to reinforce the mechanical properties of silk-based scaffolds. Better mechanical strength and suture retention were achieved in the fabric-reinforced scaffolds without sacrifice of the morphology, porous structure and cytocompatibility. Then, the stiffness and degradation behaviors could be controlled through suitable after-treatments to satisfy different requirements of various soft tissue regenerations. Considering the control and improvement of mechanical properties, the silk fabricreinforced scaffolds reported here should have clinical utility for skin tissue repairs.
Acknowledgements The authors thank Jiangsu Province Key Laboratory of Stem Cell Research for supplying the amniotic fluid stem cells. The authors thank the National Basic Research Program of China (973 Program 2013CB934400) and NSFC (21174097) for support of this work. They also thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Excellent Youth Foundation of Jiangsu Province (BK2012009), the NIH (EB002520)), Ph.D. Programs Foundation of Ministry of Education of China (201032011200009) and the Key Natural Science Foundation of the Jiangsu Higher Education Institutions of China (11KGA430002) for support of this work.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 09.026.
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Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Woven silk fabric-reinforced silk nanofibrous scaffolds for regenerating load-bearing soft tissues F. Han a,b, S. Liu a,1, X. Liu a,1, Y. Pei a, S. Bai a, H. Zhao a, Q. Lu a,⇑, F. Ma b,⇑, D.L. Kaplan c, H. Zhu d a
National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China Key Lab of Rubber–Plastics (QUST), Ministry of Education, College of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042, China c Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA d Research Center of Materials Science, Beijing Institute of Technology, Beijing 100081, China b
a r t i c l e
i n f o
Article history: Received 19 April 2013 Received in revised form 3 September 2013 Accepted 23 September 2013 Available online 1 October 2013 Keywords: Silk Dermal substitute Mechanical properties Scaffolds Suture retention
a b s t r a c t Although three-dimensional (3-D) porous regenerated silk scaffolds with outstanding biocompatibility, biodegradability and low inflammatory reactions have promising application in different tissue regeneration, the mechanical properties of regenerated scaffolds, especially suture retention strength, must be further improved to satisfy the requirements of clinical applications. This study presents woven silk fabric-reinforced silk nanofibrous scaffolds aimed at dermal tissue engineering. To improve the mechanical properties, silk scaffolds prepared by lyophilization were reinforced with degummed woven silk fabrics. The ultimate tensile strength, elongation at break and suture retention strength of the scaffolds were significantly improved, providing suitable mechanical properties strong enough for clinical applications. The stiffness and degradation behaviors were then further regulated by different after-treatment processes, making the scaffolds more suitable for dermal tissue regeneration. The in vitro cell culture results indicated that these scaffolds maintained their excellent biocompatibility after being reinforced with woven silk fabrics. Without sacrifice of porous structure and biocompatibility, the fabric-reinforced scaffolds with better mechanical properties could facilitate future clinical applications of silk as matrices in skin repair. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Rapid wound closure following large-surface and deep injuries protects wounds from infection and dehydration and prompts skin regeneration. By far, autologous skin graft is still the ideal approach to close or replace irreparably damaged skin. However, a lack of donor skin grafts has led to a search for alternative substitutes to replace damaged tissue [1,2]. A range of dermal substitutes have been developed for skin regeneration, including decellularized dermis from human or animals, scaffolds composed of natural and/or artificial polymers such as collagen, fibronectin, polycaprolactone and poly(lactic-co-glycolic acid) [2,3]. Besides interconnecting porous structure, good biocompatibility and biodegradation to provide suitable space and environments for cellular and tissue growth, a qualified scaffold should also provide the desired mechanical strength to bear suture tension in the implantation process and maintain the structure and functional properties of the tissue after implantation [4,5]. A strong correlation between scaffold stiffness ⇑ Corresponding authors. Tel.: +86 512 67061649 (Q. Lu), +86 532 84022950 (F. Ma). E-mail addresses: [email protected] (Q. Lu), [email protected] (F. Ma). 1 The authors contributed equally with the first author.
and cell behavior was revealed recently, which showed that scaffold stiffness influences the differentiation of mesenchymal stem cells [6–8]. These findings imply more rigorous mechanical requirements for the scaffolds used in load-bearing soft tissues that have low stiffness with high strength to undergo loading and strength in tension. However, obtaining the appropriate stiffness as well as strength in scaffolds is still a challenge in general for single natural or synthetic polymer. As a natural biomaterial, silk has been used as biomedical suture for decades. Recently, silk porous biomaterials have been applied in soft tissue regeneration, including skin, nerve, blood vessel, ligament and cartilage repair, owing to its impressive biocompatibility, biodegradation, low inflammatory reactions and high processability [9–16]. A number of methods, such as freeze-drying, salt leaching, gas forming and electrospinning, have been developed to prepare regenerated silk scaffolds with various conformations and microstructures [17–21]. All these porous silk biomaterials are biocompatible and provide favorable microenvironments for both cellular infiltration and growth in vitro and tissue regeneration in vivo. However, similar to other natural biomaterials, the regenerated silk scaffolds exhibit inferior mechanical properties, particularly for applications requiring strength in tension. Degummed silk fibers, that is, sericin-extracted
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.09.026
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fibers, have been investigated particularly for the purposes of ligament and tendon repair, because of their extraordinary strength. But cellular attachment and growth as well as ease of implantation may be limited for a fiber-only material [22–24]. Conversely, the stiffness of the degummed fibers (tensile strength 24.88 ± 4.95 MPa [25]) is also too high for some soft tissues such as skin (in vivo elastic modulus 0.13–0.66 MPa [26]), which might result in a negative influence on tissue regeneration. The special microstructure of soft tissues offers a possible template for designing scaffolds with suitable stiffness and strength. Many soft tissues can be viewed as hydrogel matrixes containing cells and reinforcing fibers [27]. Therefore, a technique that has developed significantly in the field of scaffold fabrication for tissue regeneration is the use of fiber reinforcement within cell-seeded natural biomaterial scaffold systems [4,28]. Recently, silk and collagen sponges were incorporated in knitted silk fiber scaffold to improve cell proliferation and anterior cruciate ligament reconstruction [9,29]. However, this study designed a silk fiber-reinforcing scaffold system composed mainly of silk fibers rather than porous scaffold, which might be problematic in applications such as nerve (in situ stress 0.05 MPa [30]) and dermis because of high stiffness and lack of porous structures in the system. In order to satisfy the requirements of regenerating load-bearing soft tissues, it is necessary to design a new silk fiber-reinforcing system composed mainly of porous scaffolds without the sacrifice of enough strength from fibroin fibers. Nanofiber scaffolds have advanced as tissue engineering scaffolds, because they mimic the fibrous nanostructure of native extracellular matrix (ECM) [31–33,21]. In a recent study, freezedried nanofiber silk scaffolds were prepared directly by regulating the self-assembly of fibroin in aqueous solution [34]. Based on this development, it is possible to achieve an excellent microenvironment with fibroin-based scaffolds for different soft tissue regeneration, if the strength of the scaffolds is further improved. Therefore, the long-term goal of this work is to fabricate a mechanically viable, silk-based scaffold system that is suitable for different soft tissue regeneration. In an attempt to improve the mechanical properties, the present authors investigated the effectiveness of reinforcing silk scaffold with silk woven fibers. Unlike the previous study, the fiber-reinforcing system is composed mainly of porous scaffold rather than fibers. The stiffness of the system is from fibroin porous scaffold and could be regulated by various after-treatments such as water annealing and methanol immersion. The cell compatibility was also investigated to confirm the influence of silk fibers on the biocompatibility of the fiber-reinforcing scaffold system, indicating that this scaffold is suitable for producing a dermal substitute for skin regeneration.
2. Experimental
the hood for 72 h at room temperature to concentrate the solution with controlled rate. During the concentrating process, silk fibroin self-assembled to nanofibers that would facilitate porous structure formation in following lyophilization. Then the 25–30 wt.% concentrated silk solution was obtained and finally diluted to 2–3 wt.% with deionized water. 2.2. Preparation of fiber-reinforced scaffolds Raw Bombyx mori silk fibers were used in this study. The woven fabrics were produced by the multiple-warp weaving method on a weaving machine (Longda, ASL2300, Tianjin, China) to form a plain weaving structure. Then the fabrics were boiled for 20 min in an aqueous solution of 0.02 M Na2CO3 to remove the sericin proteins. The sericin-free woven silk fabrics were placed on the bottom of a polystyrene mold and then immersed in diluted silk solution obtained from the procedure in Section 2.1. The mold filled with the silk solution was directly placed at 20 °C for 12 h and lyophilized for 48 h. After lyophilization, the final step was to induce crystallinity. Crystallinity can be induced by water annealing or methanol immersion to achieve water stability, according to published procedures [34]. Water annealing is the process in which the silk scaffolds are incubated in a humid environment for several hours to facilitate the transformation from random structure to silk I and silk II crystals. Briefly, the scaffolds were placed on a removable platform under which water was filled in a desiccator with a 25 in. Hg vacuum for 6 h. Unlike the water annealing process, the methanol annealing process could achieve more silk II formation by immersing the samples in 80% (v/v) methanol for 30 min. The changes in crystal structures in the treated scaffolds further resulted in the adjustment of mechanical properties of the scaffolds. The water-annealed and methanol-annealed fiber-reinforced scaffolds are termed WA-C and MA-C, respectively. In these fiber-reinforced systems, the weight content of woven silk fabrics was 30.6 ± 4.3%, indicating that the systems were composed mainly of silk porous scaffolds. As controls, woven fabrics and silk scaffolds without fabrics were also prepared under the same process and treated by water annealing or methanol immersion, which are termed WA-F, MA-F, WA-S, MA-S. The procedure of preparation of fiber-reinforced scaffolds is shown in Supporting Fig. S1. 2.3. Fourier transform infrared spectroscopy The structures of the silk woven fabrics and silk sponge in the combined scaffolds were analyzed by Fourier transform infrared (FTIR) spectroscopy on a NICOLET FTIR 5700 spectrometer (Thermo Scientific, FL, America) equipped with a MIRacle™ attenuated total reflection Ge crystal cell in reflection mode. Slices 1 mm thick were cut from the scaffolds with a razor blade in liquid nitrogen. For each measurement, 64 scans were coded with resolution 4 cm1, with the wave number ranging from 400 to 4000 cm1.
2.1. Preparation of silk solutions 2.4. X-ray diffraction Bombyx mori silk solutions were prepared according to a recently published procedure [35]. Cocoons were boiled for 20 min in an aqueous solution of 0.02 M Na2CO3, and then rinsed thoroughly with deionized water to extract sericin proteins. The degummed silk was dissolved in 9.3 M LiBr solution at 60 °C for 4 h, yielding a 20 wt.% solution. This solution was dialyzed against deionized water using Slide-a-Lyzer dialysis cassettes (Pierce, molecular weight cut-off 3500 D) for 72 h to remove the salt. Then the solution was centrifuged at 9000 rpm for 20 min at 4 °C to remove silk aggregations formed during the process. The final concentration of aqueous silk solution was 7 wt.%, determined by weighing the remaining solid after drying at 60 °C. This fresh silk solution was put into the oven for 24 h at 60 °C and then put into
X-ray diffraction (XRD) was also performed on samples with an X-ray diffractometer (X0 Pert-Pro MPD, PANalytical, Almelo, Holland) with Cu Ka radiation at 40 kV, 30 mA with a scanning rate of 0.6° min1. Before examination, the dried samples were pressed into sheets with a forcing compressor. 2.5. Scanning electron microscopy The surface and cross-section images of the fabrics and silkbased scaffolds were examined by scanning electron microscopy (SEM) (Model S-4800, Hitachi, Tokyo, Japan) at 3 kV, to avoid the destruction of silk structure. Before SEM examination, the dried
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scaffolds were cut with a razor blade in liquid nitrogen and then sprayed with platinum. 2.6. Mechanical testing The suture retention strengths in dry and wet states were obtained according to previously reported methods [36]. One end of a scaffold sheet sample was clamped (length 10 mm, width 5 mm, thickness 1.5 mm) and a 5–0 polyester suture (Jinhuan Company, Shanghai, China) was passed through the scaffold sheet, 2 mm from the edge. The suture was pulled at a rate of 50 mm min1 until pull out. This test was repeated five times per scaffold at different sites. Tensile mechanical testing was performed on the scaffolds in the dry and wet states using a load testing machine (Model 3366, Instron, Norwood, MA). Samples (length 50 mm, width 10 mm, thickness 1.5 mm) for tensile tests were clamped with a 30 mm inter-clamp distance and pulled at a rate of 10 mm min1 until rupture. Maximum stress and strain at rupture were measured. The Young’s modulus representing the elasticity was obtained by measuring the slope of the stress–strain curve in the elastic region. For measurement in the wet state, the samples were soaked in PBS at 37 °C for 1 h and then measured. Since the silk scaffolds could be fully saturated when immersed in water for >10 min, the samples in the present study were soaked in water for 1 h to ensure the achievement of saturated states. The analysis was repeated five times for each sample. Then the representative images were presented. 2.7. Silk degradation in vitro Water-insoluble scaffolds were incubated at 37 °C in 40 ml PBS containing 5 U ml1 protease XIV. Each solution contained an
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approximately equivalent mass (40 ± 5 mg) of silk scaffold with 30.6 ± 4.3% woven silk fabrics. At designated time points, samples were rinsed with deionized water and dried by lyophilization and prepared for mass balance assessment. The analysis was repeated five times for each sample. The morphologies after degradation were also examined using a Hitachi S-4800 SEM (Model S-4800, Hitachi, Tokyo, Japan).
2.8. Cell compatibility Homo sapiens dermal fibroblasts Hs 865.Sk cells obtained from ATCC (American Type Culture Collection) were used to assess the cytocompatibility of the scaffolds. Cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS), 1% IU ml1 streptomycin–penicillin (all from Invitrogen, Carlsbad, CA). Amniotic fluid-derived stem cells, with a capacity to differentiate into multiple cells and avoid ethical concerns associated with embryonic stem cells, were studied to assess the cytocompatibility of the scaffolds. Samples of amniotic fluid were obtained from the First Affiliated Hospital of Soochew University (Suzhou, Jiangsu, China) following routine amniocentesis carried out on pregnant women at 15–35 weeks of gestation. All the procedures were performed following the guidelines established by the First Affiliated Hospital of Soochew University and the First Affiliated Hospital of Soochew University Ethics Committee; written consent to use the amniotic fluid for research purposes was obtained from each woman. The isolation of human amniotic fluid-derived stem cells has been described previously [37]. Amniotic fluid-derived stem cells were cultured in a-Modified Eagle Medium supplemented with 10% FBS, 1% IU ml1 streptomycin– penicillin (all from Invitrogen, Carlsbad, CA) and growth factor: Basal, C Frozen Supplement (Irvine Scientific, Santa Ana, CA). The
Fig. 1. Microstructures of silk scaffolds and woven silk fabric-reinforced scaffolds: (a) SEM image of silk scaffolds; (b) SEM image of degummed woven fabrics; (c) microstructure of silk scaffolds near woven fabrics (arrows indicate the woven fabrics); and (d) microstructure of silk scaffolds away from woven fabrics.
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medium was replaced every 3 days, and the cultures were maintained in a humidified incubator at 37 °C and 5% CO2. Different methods, including steam, ethanol and gamma radiation, have been used to sterilize silk-based biomaterials [38]. However, considering that random and silk I structures of the water-annealed scaffolds could transform into silk II structure in steam or ethanol sterilization processes, gamma radiation was chose to sterilize the samples. The scaffolds (thickness 2 mm) were punched into small disks with diameter 8 mm and then sterilized by c radiation at room temperature, with 25 kGy from a 60Co Gammacell 220 facility, which had a dose rate of 2 kGy h1. After pre-conditioning with the culture medium overnight, the samples were transferred into a non-tissue culture treated 96-well plate and seeded with Hs 865.Sk cells at a density of 1.6 105 well1. Three hours after cell seeding, samples were transferred into a non-tissue culture-treated 6-well plates, and 5 ml complete culture medium was added to each well for up to the indicated time points. 2.9. DNA content To study cell proliferation on the scaffolds, samples were harvested at the indicated time point (from day 1 to day 12), and digested with proteinase K buffer solution for 16 h at 56 °C, as described previously [39]. The DNA content was determined using the Quant-iTTM PicoGreen dsDNA assay, following the protocols of the manufacturer (Invitrogen, Carlsbad, CA). Samples (n = 5) were measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm, using a BioTec Synergy 4 spectrofluorometer (BioTec, Winooski, UK). The amount of DNA was calculated by interpolation from a standard curve prepared with lambda DNA in 10 10–3 M Tris–HCl (pH 7.4), 5 10–3 M NaCl, 0.1 10–3 M EDTA over a range of concentrations.
3. Result and discussion 3.1. Morphology of fabric-reinforced silk scaffolds The morphology changes of silk in the treated solution are shown in Fig. S2. Through a slowly concentrating process, the nanoparticles were transformed into nanofibers in silk solution (Fig. S2A and B), which restrained the formation of lamellar structures [34] and then achieved excellent porous structures after lyophilization (Fig. 1A). The ECM provides a framework for scaffold design in which nanoscale and microscale dimensions of the physical structures are important, including nanofibrous architectures as well as specific porous structures [31–33,21]. Previous studies have confirmed that silk nanofiber formation could further improve the biocompatibility of silk scaffolds, which would provide better microenvironments for soft tissue regeneration. However, the fragile mechanical properties of the regenerated silk scaffolds restrained its clinical applications in tissue regeneration [17–21]. Therefore, the woven fabrics (Fig. 1B) were introduced into the scaffolds to afford enough mechanical strength for clinical applications in soft tissue regeneration (Fig. 1C). The degummed woven fabrics had a smooth surface after the extraction of sericin (Fig. 1B). Compared with silk fibroin scaffolds, the fabric-reinforced scaffolds had a similar interconnected porous structure, with pore
2.10. Cell viability and morphology The morphology of Hs 865.Sk cells seeded on the scaffolds was examined by confocal microscopy. Briefly, the cell-seeded scaffolds were washed three times with PBS (pH 7.4) and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 30 min, followed by further washing. The cells were permeabilized using 0.1% Triton X-100 for 5 min and incubated with FITC-phalloidin (SigmaAldrich, St. Louis, MO) for 20 min at room temperature, followed by PBS washing and finally staining with DAPI (Sigma-Aldrich, St. Louis, Mo) for 1 min. Representative fluorescence images (10) of stained samples were obtained by confocal laser scanning microscopy (CLSM, Olympus FV10 inverted microscope, Nagano, Japan) with excitation/emission at 358/461 nm and 494/518 nm. The images of the scaffolds were captured from surface deep to 100 lm at increments of 10 lm, respectively. The cell morphology on the scaffolds was confirmed by SEM. After harvest, the cellseeded scaffolds were washed three times with PBS and fixed in 4% paraformaldehyde at room temperature, and then the scaffolds were washed three times with PBS again. Fixed samples were dehydrated through a gradient of alcohol (50%, 70%, 80%, 90%, 100%, 100%) followed by lyophilization. Specimens were examined using a Hitachi Model S-4800 SEM (Hitachi, Tokyo, Japan). 2.11. Statistical methods All statistical analyses were performed using SPSS v.16.0 software. Comparison of the mean values of the data sets was performed using two-way ANOVA. Measures are presented as means ± standard deviation, unless otherwise specified, and P < 0.05 was considered significant.
Fig. 2. (A) FTIR spectra and (B) XRD data for silk fabrics and scaffolds after different treatments: (a) degummed silk fabrics; (b) water-annealed silk scaffolds; (c) waterannealed fabric-reinforced scaffolds; (d) methanol-annealed silk scaffolds; and (e) methanol-annealed fabric-reinforced scaffolds.
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size ranging from 200 to 300 lm, indicating that the addition of silk woven fabrics did not have a negative effect on porous structure formation (Fig. 1C and D). The scaffolds were then treated by water or methanol annealing to achieve water stability without significant porous structural changes (data not shown). Importantly, the weight ratio of the fabrics was significantly decreased compared with that of previous silk fiber-reinforced sponges used in ligament reconstruction [40], making it possible to be used in soft tissue regeneration. Although the weight ratio of fabrics was 30% in the scaffolds, the fabrics were only located near the surface of the scaffolds. The woven fabric was a thin lamellar, meaning
Fig. 3. Suture retention strengths of silk scaffolds: WA-S, water-annealed silk scaffolds; MA-S, methanol-annealed silk scaffolds; WA-C, water-annealed fabricreinforced scaffolds; and MA-C, methanol-annealed fabric-reinforced scaffolds (⁄P<0.05).
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that most part of the scaffold was porous sponge. The special structure might reduce the influence of stiff fabrics on cell behaviors. 3.2. Structural analysis Two major structural models, termed silk I and silk II, exist in silk fibroin. Alpha-helix dominated the silk I structure existing within the silkworm gland just before spinning. Silk II is the insoluble anti-parallel beta-sheet crystal conformation, which forms after the spinning of silk fibers from the spinneret of the silkworm [41]. The physical properties of silk fibers, such as mechanical, thermal, optical and dielectric properties, vary, depending on the structure of the silk and on the content of beta-sheet crystals. Several methods are used to induce beta sheet crystallization in silk biomaterials, such as methanol annealing and steam-autoclaving [41]. The structures of the scaffolds were determined by FTIR (Fig. 2A). The IR spectral region within 1700–1500 cm1 is assigned to absorption by the peptide chains of amide I (1700–1600 cm1) and amide II (1600–1500 cm1), which have usually been used for the analysis of secondary structures of silk fibroin. The peaks at 1610–1630 cm1 (amide I) and 1510–1520 cm1 (amide II) are characteristic of silk II secondary structure, while the absorptions at 1648–1654 cm1 (amide I) and 1535–1542 cm1 (amide II) are indicative of silk I conformation [35,42]. Similarly to many previous studies [23,24,28,29], the silk fabrics are mainly composed of stable silk II structure without conformational changes after water annealing or methanol annealing treatments. In order to avoid the overlap between silk fabrics and porous sponges, the sponges were torn from the scaffolds and measured independently before and after the treatments. The spectra of WA-S and the silk sponge part of WA-C showed strong peaks at 1650 cm1 and 1535 cm1, which represented a typical silk I structure. After treatment with 80% methanol for 30 min, the strength of the peak at 1625 cm1 significantly increased, implying silk II formation. These results
Fig. 4. Stress–strain curves of samples in the dry and wet states: (A) water-annealed samples in dry state; (B) water-annealed samples in wet state; (C) methanol-annealed samples in dry state; (D) methanol-annealed samples in wet state; (a) silk scaffolds; (b) fabric-reinforced silk scaffolds and (c) woven fabrics alone.
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3.3. Mechanical properties
Table 1 Mechanical properties of silk scaffolds in dry and wet states. Scaffolds
Tensile strength (MPa)
Strain at break (%)
Modulus (MPa)
Dry-WA-F Dry-MA-F Dry-WA-S Dry-MA-S Dry-WA-C Dry-MA-C Wet-WA-F Wet-MA-F Wet-WA-S Wet-MA-S Wet-WA-C Wet-MA-C
2.29 ± 0.04 2.39 ± 0.02 0.11 ± 0.03 0.13 ± 0.04 2.97 ± 0.57 3.39 ± 0.63 1.70 ± 0.01 1.74 ± 0.08 0.04 ± 0.003 0.05 ± 0.01 2.01 ± 0.16 2.11 ± 0.11
15.64 ± 1.23 16.09 ± 1.36 6.33 ± 1.48 6.73 ± 2.22 24.0 ± 2.41 29.0 ± 1.57 20.92 ± 1.64 20.89 ± 1.33 20.87 ± 1.94 20.27 ± 2.05 35.63 ± 0.68 41.57 ± 2.50
1.86 ± 0.19 2.51 ± 0.23 0.66 ± 0.20 0.77 ± 0.17 4.05 ± 0.48 4.40 ± 0.15 0.37 ± 0.08 0.62 ± 0.10 0.22 ± 0.06 0.53 ± 0.03 0.34 ± 0.01 0.60 ± 0.05
indicated that the silk fabrics had no significant inhibition on conformational transition in different treatment processes. The structural changes were confirmed by XRD results (Fig. 2B). Silk fabrics showed silk II crystal structure, with a sharp peak at 20.2° (II). The WA-S and sponge part of WA-C were characterized by diffraction peaks at 2h values of 12.2° (I), 19.7° (I), 24.7° (I) and 28.2° (I), indicating silk I structure [43]. After methanol annealing, the silk I peaks at 12.2°, 19.7°, 28.2° disappeared, and the strength of the silk II peak at 20.2° increased, indicating silk II formation in MA-S and the sponge part of MA-C. The structural study implied that the secondary structures could be regulated by different treatments, which resulted in the changes in mechanical properties and degradation behaviors of the fabric-reinforced scaffolds.
Suture retention is a critical factor in the design of scaffolds used in different tissue regeneration, as it directly relates to the success of the implant. Various tissues, experiencing different load ranges, have different requirements for suture retention strength. The suture retention strength of regenerated silk fibroin scaffolds is usually inferior to that of requirements in clinical applications, which even results in the failure of an implant. In this study, woven silk fabrics were used to withstand suturing forces. The suture retention strengths of the WA-C scaffolds and MA-C scaffolds were 3.7 ± 0.6 N and 3.7 ± 0.3 N in the dry state, and then slightly decreased to 3.2 ± 0.5 N and 3.1 ± 0.8 N in the wet state. The retention strengths showed no significant difference between water annealing and methanol annealing treatments. But the fabric-reinforced scaffolds showed better retention strength than the scaffolds without fabric (<0.6 N in dry and wet states), confirming the critical function of woven fabrics for withstanding tensile forces during suturing (Fig 3). Although the suture retention strength of fabricreinforced scaffolds is still significantly inferior to reticular dermis (46.2 N) and the decellularized dermis ECM (47.0 N) [44], considering that the generally accepted adequate suture retention strength for small blood vessel in surgery is >1.8 N [36], the present authors’ fabric-reinforced scaffolds might be acceptable for clinical dermis implants. Further study is necessary to improve the suture retention strength of silk-based scaffolds through regulating the conformations and nanostructures of silk. Besides suture retention strength, the tensile properties of silk scaffolds also improved after the addition of silk woven fabrics
Fig. 5. (A) Degradation behaviors of silk scaffolds in 5 U ml1 protease XIV solution. (a) WA-S, water annealed silk scaffolds; (b) WA-C, water annealed fabric-reinforced silk scaffolds; (c) MA-S, methanol annealed silk scaffolds; (d) MA-C, methanol annealed fabric-reinforced silk scaffolds; (e) WA-F, water annealed woven fabrics; (f) MA-F, methanol annealed woven fabrics. (B) SEM images of fabric-reinforced silk scaffolds incubated in protease XIV solution at 37 °C for 2 h: (a) WA-C; (b) MA-C.
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(Fig. 4 and Table 1). The ultimate tensile strengths (UTS) of WA-C and MA-C scaffolds were 3–4 MPa, above twenty times the strength of the corresponding scaffolds (WA-S, 110 KPa; MA-S, 130 KPa) in the dry state. The tensile strength of the hydrated fabric-reinforced scaffolds decreased slightly to 2.0–2.1 MPa, which was still significantly higher than that of scaffolds without woven fabric in the wet state (WA-S, 40 KPa; MA-S, 50 KPa). The higher elongations at break were also achieved for WA-C and MA-C scaffolds, which were >20% in the dry state and then further increased to >35% in the wet state. In addition, the mechanical properties of the woven silk without porous sponges were also investigated. The tensile strengths and elongations at break were 2.29–2.39 MPa and 15.64–16.09% in the dry state and then changed to 1.70–1.74 MPa and 20.89–20.92% in the wet state (Table 1), which was also slightly inferior to the fabric-reinforced scaffolds. The
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results further confirmed that better mechanical properties could be achieved through the fabric-reinforcing method. Scaffold stiffness is an important mediator of cell behavior to regulate cell signaling and, eventually, the formation and maintenance of tissues [45]. Many soft tissues are composite with collagen fibrils reinforcing hydrogel to form soft but strong matrix. In the present fabric-reinforcing scaffolds, woven fabrics afforded remarkably high strength, while silk sponge maintained low stiffness to simulate the microenvironment in vivo. Table 1 shows that the modulus of fabric-reinforced scaffolds in the wet state was similar to the corresponding scaffolds without fabrics and could be further regulated by different after-treatments. For example, the modulus of WA-C was 0.34 MPa, which changed to 0.60 MPa for MA-C in the wet state. The results indicated that the stiffness of the fabric-reinforcing scaffolds could be modulated with various
Fig. 6. Confocal microscopy of amniotic fluid-derived stem cells cultured on different scaffolds at day 1 and day 12: (A1) water-annealed silk scaffolds (WA-S), day 1; (A2, A3) water-annealed silk scaffolds (WA-S), day 12; (B1) water-annealed fabric-reinforced silk scaffolds (WA-C), day 1; (B2, B3) water-annealed fabric-reinforced silk scaffolds (WA-C), day 12; (C1) methanol-annealed silk scaffolds (MA-S), day 1; (C2, C3) methanol-annealed silk scaffolds (MA-S), day 12; (D1) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 1; (D2, D3) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 12. The blue color (DAPI) represents silk scaffolds and cell nucleus, while the green color (FITC labeled phalloidin) represents actin cytoskeleton.
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after-treatments to satisfy the specific requirements of different tissue regenerations without sacrifice of mechanical strength. The mechanical properties of skin in vivo and in vitro have been investigated by different groups. Although the results are generally different, owing to the changes of specimen location and test methods, recent study showed that the mean tensile strength, failure strain and elastic modulus of in vitro human skin from the back were 21.6 MPa, 54% and 83.3 MPa, respectively [46]. These mechanical properties were significantly superior to those of the present fabric-reinforcing scaffolds, implying that the scaffolds cannot replace the real dermis. However, the mechanical properties of skin in vitro were measured in the high-strain range, while the real skin maintained its function in the low-strain range. Therefore, it is more feasible to compare the mechanical properties of the fabric-reinforcing scaffolds and the skin in vivo. Interestingly, the moduli of WA-C and MA-C were 0.34 MPa and 0.60 MPa in the wet state, which were similar to the modulus of the skin in vivo tension test (0.13–0.66 MPa) [26]. Considering the influence of scaffold stiffness on regulating cell behavior and tissue regeneration, the present scaffolds may provide a suitable microenvironment for skin regeneration.
observed on the scaffolds. After day 12, cells increased and formed a continuous monolayer on the surface of the porous walls. Confocal microscopy and DNA content confirmed the cytocompatibility of the scaffolds (Figs. 7 and 8). Cells proliferated significantly and interacted to form aggregates at day 12. The cytocompatibility of the fabric-reinforced scaffolds showed no significant difference compared with that of scaffolds without fabrics, confirming that reinforcing silk scaffolds with silk fabric is a feasible way to improve the mechanical properties of silk-based scaffolds without sacrifice of cytocompatibility. Moreover, the cell compatibility assay was repeated using human dermal fibroblasts Hs 865.Sk cells and the same results were found (Figs. S4–S6). Considering the possible application of the scaffolds in skin regeneration, human dermal fibroblast cells were also cultured on the scaffolds, a common and valid way to assess the biocompatibility of biomaterials in skin repair, to assess the feasibility of the scaffolds. Similar to stem cells, human dermal fibroblasts Hs 865.Sk cells also showed
3.4. Degradation After incubation in protease XIV solution (5 U ml1) at 37 °C for 24 h, WA-S scaffold with a low content of b-sheet was almost completely degraded, while only silk fabrics remained in the WA-C scaffold, implying inconsiderable influence of the fabrics on the degradation behavior of the sponge part. Following the increase in b-sheet content after methanol treatment, the weight losses were 52% for MA-S and 39% for MA-C (Fig. 5A). The results indicated that the degradation behaviors of the scaffolds could be regulated through controlling crystal structure by different aftertreatments. Considering little degradation of silk fabrics and the content of fabrics in MA-C scaffolds (30 wt.%), the degradation ratio of the porous sponge part was 50% in MA-C, similar to that of MA-S, which confirmed the minimal effect of the fabrics. The morphology changes of the scaffolds in the degradation process were also investigated (Fig. 5B). The nanofibrous structure appeared on the surface of the macropore walls following degradation, which might promote cell adhesion, growth and migration in vitro or in vivo [34]. The mechanical properties of the fabric-reinforced scaffolds and the woven fabric in the degradation process were also measured. After the scaffold part had almost degraded, the mechanical properties of the fabric-reinforced scaffolds slightly decreased and were similar to those of the woven fabric after the same degradation treatment. The results supported the theory that the woven fabric endured the main mechanical tension in the fabric-reinforced scaffolds. 3.5. Cell compatibility Dermal tissue engineered constructs can be achieved based on these scaffolds. The present authors’ further work involves inducing targeted differentiation of stem cells on these scaffolds. Amniotic fluid stem cells show multilineage differentiation potential into various tissue types such as skin, cartilage, cardiac tissue, nerves, muscle and bone. Moreover, compared with other stem cell sources, amniotic fluid-derived stem cells possess immunosuppressive activity [47]. Therefore, in the present study, amniotic fluid stem cells were chosen to analyze the biocompatibility of the scaffolds. Amniotic fluid-derived stem cells were cultured in the scaffolds with and without silk fabrics, respectively. All the scaffolds showed excellent cytocompatibility (Fig. 6). The amniotic fluid-derived stem cells adhered and grew well on the surfaces of the scaffolds from day 1 to day 12. At day 1, only a few cells were
Fig. 7. SEM images of Hs 865.Sk cells cultivated on different scaffolds at day 1 and day 12: (A) water-annealed silk scaffolds (WA-S), day 1; (B) water-annealed silk scaffolds (WA-S), day 12; (C) water-annealed fabric-reinforced silk scaffolds (WA-C), day 1; (D) water-annealed fabric-reinforced silk scaffolds (WA-C), day 12; (E) methanolannealed silk scaffolds (MA-S), day 1; (F) methanol-annealed silk scaffolds (MA-S), day 12; (G) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 1; (H) methanol-annealed fabric-reinforced silk scaffolds (MA-C), day 12.
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Appendix B. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1 and 6, are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi: http://dx.doi.org/10.1016/ j.actbio.2013.09.026.
References
Fig. 8. Amniotic fluid-derived stem cells proliferation on silk scaffolds: WA-S, water annealed silk scaffolds; WA-C, water annealed fabric-reinforced silk scaffolds; MAS, methanol annealed silk scaffolds; and MA-C, methanol annealed fabric-reinforced silk scaffolds. Error bars represent mean ± standard deviation with n = 5.
excellent growth and proliferation behaviors on the scaffolds (Figs. S4–S6). Although it is important to study further the differentiation behavior of the amniotic cells into skin cell types on the fabric-reinforced scaffolds, the present results have proved that the scaffolds have good biocompatibility for skin regeneration, which is critical for future clinical applications. Further study in the near future will be focused on the directed differentiation of amniotic fluid stem cells into skin cell types by adding certain factors to provide better microenvironments for skin regeneration.
4. Conclusions Degummed silk fabric was used to reinforce the mechanical properties of silk-based scaffolds. Better mechanical strength and suture retention were achieved in the fabric-reinforced scaffolds without sacrifice of the morphology, porous structure and cytocompatibility. Then, the stiffness and degradation behaviors could be controlled through suitable after-treatments to satisfy different requirements of various soft tissue regenerations. Considering the control and improvement of mechanical properties, the silk fabricreinforced scaffolds reported here should have clinical utility for skin tissue repairs.
Acknowledgements The authors thank Jiangsu Province Key Laboratory of Stem Cell Research for supplying the amniotic fluid stem cells. The authors thank the National Basic Research Program of China (973 Program 2013CB934400) and NSFC (21174097) for support of this work. They also thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Excellent Youth Foundation of Jiangsu Province (BK2012009), the NIH (EB002520)), Ph.D. Programs Foundation of Ministry of Education of China (201032011200009) and the Key Natural Science Foundation of the Jiangsu Higher Education Institutions of China (11KGA430002) for support of this work.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 09.026.
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