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Structural Mimetic Silk Fiber-Reinforced Composite Scaffolds Using Multi-Angle Fibers Gang Li, Jian Liu, Zhaozhu Zheng, Xiaoqin Wang,* David L. Kaplan* The fabrication of structural mimetic scaffolds reinforced with multi-angle silk fibers is described. Degummed silk fibers with a parallel arrangement of the fibers in a planar format were overlapped with successive layers organized at 08, 308, 608 and 908, respectively. The overlapped silk fiber layers were coated with silk solution (6 wt%) containing sodium dodecyl sulfate (SDS). The morphology, mechanical properties, structure and biocompatibility of the scaffolds were investigated. The mechanical properties of the scaffolds (tensile and burst) were characterized based on the angles of the fibers. Layers with an overlapping angle at 308 exhibited better mechanical performance (18 MPa) than the other groups. The results of Fourier Transform (FT) IR Spectroscopy (FT-IR) and X-ray Differentiation (XRD) analyses indicated that the presence of degummed silk fibers with different angles did not significantly impact secondary structure or crystallization of the fiber reinforced scaffolds. The attachment and growth of a human fibroblast cell line (HS-865-SK) on the reinforced scaffolds supported good cell compatibility. These new scaffolds have potential applications in tissue repairs where superior mechanical strength and cell compatibility are important.
In recent decades, fiber-reinforced composites (FRCs) have emerged as intriguing substitutes for plastics and metals in industrial applications[1] and represent successful biomaterials for biomedical applications.[2] Unlike homogeneous and isotropic materials, FRCs provide the possibility of designing materials with enhanced and tailored mechanical properties by selecting the material constituents (basic
G. Li, J. Liu, Z. Zheng, X. Wang National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, P. R. China E-mail: [email protected] D. L. Kaplan Department of Biomedical Engineering, Tufts University, 4 Colby St., Room 153, Medford, MA 02155, USA E-mail: [email protected]
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
matrix and reinforcing components, such as fibers) and setting their volume ratios.[1] Mechanical properties for structural engineering applications can be improved if the reinforcing fibers are properly blended with the matrix materials.[3–6] A critical goal for bioengineering functional and loadbearing tissues is the recapitulation of the robust mechanical properties intrinsic to the in vivo structures.[7] The use of FRCs in the preparation of medical materials and devices for soft and hard tissue replacement requires a wide selection range for the mechanical properties, as well as maintenance of biocompatibility.[8] Of the broad spectrum of biomimetic products, FRCs are often used for mechanically robust scaffolds consisting of a polymer matrix imbedded with high-strength fibers,[2,9] as well as biocompatible fibers, including silk fibers.[10] Compared to other fibers, silks consist of proteins produced naturally by silkworms and spiders, and are known as biocompatible, biodegradable, and extraordinarily robust biomaterials that are frequently
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1. Introduction
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utilized in biomaterial composites.[11–15] To produce robust matrices, a variety of functional inorganic nanostructures have been integrated with silk materials.[5,11,16,17] Yuan et al. reported a silk composite in which uniaxially-aligned and continuous-typed Bombyx mori silk fibers were embedded in a matrix of silk fibroin via a solution casting process. Compared to the individual material components, the overall mechanical properties as well as the thermal stability of the silk composites were significantly improved.[18,19] A typical application of composite materials where robust mechanics is required is for meniscus repair. Previous studies have used a variety of biodegradable scaffolds with or without the incorporation of stem cells, but the results have not been sufficient for clinical applications.[20] In order to develop mechanically robust scaffolds for meniscus repair, understanding and mimicking the complex internal architecture and function of the meniscus are critical.[21] The human meniscus is composed of an extracellular matrix where the organization and architecture of collagen fibers and proteoglycans are complex, conferring tissue-specific biomechanical characteristics. A longitudinal section through the meniscus shows that collagen fibers are arranged in three zones; superficial, lamellar and deep. The orientations of collagen fibers are of three types; circumferential, radial and random. Circumferential fibers are predominant and occupy the deep zone. Radial and random fibers are found in the lamellar zone and random fibers are scattered in the superficial zone.[22] In the present study, structural mimetic scaffolds reinforced with multi-angle silk fibers were fabricated and characterized in terms of mechanical strength, structure and cell interactions. The results indicated that silk fibers superimposed at defined angles (308 and 608) in the scaffolds significantly enhanced mechanical strength when compared to the scaffolds alone or the scaffolds with silk fibers superimposed at 08 and 908 angles. The fiber reinforcement technique and mechanisms elucidated in
this study can also be expanded to other mechanical strength-demanding tissue regeneration needs.
2. Experimental Section 2.1. Materials Degummed silk fibers were purchased from Xiehe Silk Corporation (Zhejiang, China). Lithium bromide was supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glycerol was provided by Shanghai Reagent Factory. Human dermal fibroblasts (Hs 865.Sk cells) were obtained from ATCC (American Type Culture Collection, Maryland, USA). Cell culture media were purchased from Wisent Corporation (Montreal, Canada). Other reagents were analytical grade.
2.2. Preparation of Multi-Angle Silk Fiber Reinforced Scaffolds (MSFRSs) The silk fibers were degummed by boiling in 0.02 m Na2CO3 for 30 min to remove sericin contaminants, as reported previously.[20] The degummed silk fibers (25%, w/v) were dissolved in 9.3 m LiBr solution at 60 8C for 4 h.[20] The mixing solution was dialyzed against pure water for 36 h to remove the lithium bromide. The solution was centrifuged at 12 000 g for 20 min at 4 8C to remove the insoluble fibrous debris and silk aggregates. Silk fibroin solution was then blended with 30 wt% glycerol prior to material processing to increase tenacity and surface smoothness of the regenerated silk fibroin scaffolds.[23] Double-sided tape was adhered onto the surface of a roller (diameter: 15 mm, length: 60 mm) and silk fibers were uniformly wound on the roller to form a single layer at a speed of 7 r min1. Along the axial direction, the double-sided tape was cut into two halves and removed from the roller, so that a fiber layer with parallel fiber arrangements was produced. Subsequently, the fiber layers were overlapped with different angles 08, 308, 608 and 908 respectively (Figure 1). The overlapped fiber layers were treated with 10% (w/v) sodium dodecyl sulfate (SDS) to increase cohesion between the fiber surfaces and the continuous silk fibroin matrix to improve mechanical performance. These laminates were then soaked in 1.5N mL of silk fibroin solution (6%, w/v), where N
Figure 1. Diagram of sampling approach (a) and different organizations of the fiber layers (b-e): 08, 308, 608 and 908, respectively.
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represents the number of layers of silk fibers with parallel arrangement. The samples were dried in the fume hood for 6 h, and subjected to the formation of a second layer of fibers. The process was repeated to obtain the laminates with different orientations of the constituent layers.
2.3. Characterization of MSFRSs 2.3.1. Morphology In order to compare the surface morphology of the MSFRSs before and after being treated with SDS, the samples were examined using a Scanning Electron Microscopy (SEM) (Hitachi S-4800, Tokyo, Japan). All specimens were sprayed with gold prior to imaging and observed at a voltage of 3 kV.
2.3.2. Mechanical Properties Tensile properties (samples shape and size: rectangle, 5010 mm) were examined in the hydrated state in three orientations (Figure 2a) using a load testing instrument (Model, Instron-5565, Norwood, MA) according to China standard GB/T 1040-2006. The testing conditions used included: tensile gap 28 mm; speed 20 mm min1 until rupture (T ¼ 25 8C, relative humidity (RH): 65%). The maximum strain and stress at rupture were observed and the Young’s modulus was calculated according to the slope of the stress-strain curve within the elastic region.[24,25] The burst properties of the samples (samples shape and size: round, diameter 100 2 mm) were examined for the composite materials in the hydrated state using a load testing instrument (Model: Instron3366, Norwood, MA). The measurement conditions: burst speed 20 mm min1 until rupture. The displacement-load curves were obtained for samples immersed in phosphate-buffered saline (PBS; pH ¼ 7.4) for 24 h before measurement. The measurements were repeated five times for each sample under standard testing environment (T ¼ 25 8C, RH ¼ 65%). Representative images are presented.
2.3.3. Structural Analysis The secondary structure of the MSFRSs was examined using Fourier Transform (FT) IR Spectroscopy with a MIRacleTM attenuated total reflection (ATR) Ge crystal cell in reflection mode. FT-IR wave
numbers were set from 4000 to 400 cm1 during 32 scans, with 2 cm1 resolution (Nicolet 5700, Thermo Electron Corp, Waltham, MA). The major vibration bands were attributed to specific chemical groups. The internal crystalline structure of the MSFRSs was examined via an X-ray diffractometer (X’Pert-Pro MPD, PANalytical BV, Almelo, Netherlands) using a CuKa radiation source. Two dimensional X-ray diffraction patterns were obtained at the following irradiation settings: Cu, Ka, wavelength 0.154 nm, range 58–608, 40 kV and 35 mA at a scanning rate of 58 min1.
2.4. Cell Culture and Seeding HS-865-SK cells were placed in 6-well plates at a density of 1.2 106 cells per well with 2.5 mL medium and maintained in high glucose Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Wisent Corporation, Canada) and 1% antibiotic/antimycotic at 37.0 0.2 8C in a humidified air incubator containing 5% CO2. The number of cells was counted using Cellometer Auto T4 (Nexcelom Bioscience LLC, USA). The scaffolds samples with 608 and 908 silk fiber compositions were selected and immersed in deionized water for 24 h before cell culture. Then a round-shaped sample (diameter: 15 mm, thickness: 0.15 mm) was cut from the scaffolds and placed on the bottom of 6well plates. Prior to seeding with cells, the samples were sterilized under 60Co radiation at 18 kGy (Irradiation Technology Institute of Soochow University, China). A total of 1.2 106 cells per well with 2.5 mL medium was added into the wells and incubated with the materials for 7 d.
2.5. Compatibility of MSFRSs 2.5.1. Cell Attachment and Proliferation Cells were cultured in the plate wells in which silk scaffolds (608 and 908 silk fibers) were either floated in the medium or fixed to the bottom of the wells. Plain silk membranes that were prepared in the absence of silk fibers served as controls. Cell attachment and proliferation at different time points were investigated. At each time point, three parallel samples in a testing group were taken out and subjected to measurements.
Figure 2. SEM images of fracture cross sectional views of MSFRSs, (a) MSFRS without treatment with SDS, (b) MSFRS treated with SDS.
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2.5.2. Cell Morphology Cell morphology and proliferation of each laminate group were observed using an Olympus phase contrast microscope TH4 200 (Osaka, Japan).
2.5.3. Cell Viability
surface hydrophilicity of the silk fibers and avoided bubbles formation during the blending process, thus providing higher contacting surface areas between the fibers and the continuous silk phase.[26,27]
3.2. Mechanical Properties
Methylthiazol tetrazolium (MTT) assay was conducted to determine cell viability in the presence of silk fiber-reinforced scaffolds. For viability, the cells were harvested, trypsinized and washed at 6, 24, 48 and 72 h intervals after seeding and then re-suspended gently in 1 mL PBS consisting of 10 mL of 12 mm MTT and 100 ml of SDS-HCl solution (fresh and filtered) for 12 h at 25 8C for staining. Absorbance was measured at 570 nm using a microplate reader (Model Synergy 2, Bio-Tek, Vermont, USA). All samples were run in triplicate.
2.6. Statistical Analysis Each experiment was repeated three times and all data sets are expressed in terms of mean and standard deviations. Statistical analysis was performed using one-way analysis of variance (ANOVA). The statistical difference between two groups of data was considered to be significant when P < 0.05.
3. Results and Discussion 3.1. Morphology of the MSFRSs Treated with SDS The morphologies of the MSFRSs treated with SDS before and after tensile measurement (method described above) are shown in Figure 2a. The cross-view of the fracture section during tensile measurement of the material without SDS treatment was rough and uneven, and moreover, part of the fibers pulled out of the sample. The results indicated a lack of cohesion between the fiber surface and the continuous silk fibroin matrix, which weakened mechanical performance. SDS is a commonly used surface active agent having both hydrophilic and hydrophobic groups, and has been used to improve the surface hydrophilicity of textiles due to its capabilities of emulsification, dispersion and solubilization.[23] In order to improve the mechanical properties of MSFRSs, prior to blending with SF solution, the silk fiber layers were treated by 10% (w/v) SDS for 0.5 h. The cross section view of the fracture surface of the material after the SDS treatment showed improved integration with few silk fibers being pulled out (Figure 2b). Thus, pre-treatment of the silk fibers with SDS facilitated material cohesion, giving rise to improved mechanical properties when compared to the untreated materials. The stress values of 608 sample before (and after) the treatment under axial, inclined and horizontal directions were 8.2 1.15 (9.7 1.65), 7.1 0.95 (10.2 1.05) and 7.2 1.02 (10.5 1.01) MPa, respectively. The reason is that the SDS treatment presumably increased
3.2.1. Tensile Tests The tensile stress and strain of the MSFRSs in the hydrated state were studied under axial, inclined and horizontal directions, respectively. As shown in Figure 3, the scaffolds with 08 overlapped fiber laminates exhibited a tensile stress above 40 MPa when the samples were measured under axial direction, much higher than other samples being tested (5–15 MPa). However, these samples showed the lowest isotropic tensile stress among all the samples tested. For example, the tensile stress in the inclined direction was only above 0.3 MPa, indicating that there was almost no tensile strength in the horizontal direction. This is mainly because there were few single fibers along the axial direction bearing continuous load. In contrast, the tensile stress of 308 and 608 overlapped samples under three directions was isotropic (10– 20 MPa). The 908 overlapped sample possessed high axial and horizontal tensile strength but low inclined tensile strength (3.3 MPa). In this case, the tensile behaviors were similar to that of the 08 overlapped sample, i.e., few continuous fibers were directly held by the two clamping ends on the instrument, and the fibers in the horizontal direction of the sample did not bear the tensile force. The plain SF membrane showed relatively high tensile strain (approximately 100%) due to its flexibility, but low tensile stress (3 MPa), and there was no significant difference between the strength values measured in all directions. The data suggested that embedding silk fibers in SF solution enhanced the strength of the SF laminate materials and the angle with regard to the fiber layers overlapped to each other had significant impact on the mechanical properties of the SF laminates. 3.2.2. Burst Tests The burst strength of various MSFRSs in the hydrated state was studied. As shown in Figure 4, the scaffolds with 308 overlapped fiber layers showed the highest burst strength above 160 N. For example, the burst strength of 08, 608 and 908 overlapped samples and the plain SF membranes were 15, 100, 75 and 20 N, respectively. When the material was subjected to burst pressure, it stretched in different directions and the region showing the lowest deformation strength was prone to crack first. Due to the uneven structural composition, the 08 overlapped samples showed the lowest tensile properties (see above) and burst strength
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Figure 3. The tensile curves and comparison of hydrated MSFRSs, a, b and c are tensile curves and stress comparisons of the samples under axial, inclined and horizontal directions, respectively. d is the results of a-c; A-E represent samples with multi-angle overlapping fiber layers at 08, 308, 608, 908 and the plain SF membrane, respectively.
under inclined and horizontal directions and linear cracks were observed (Figure 5B(a)). Therefore, these materials were prone to crack at the fiber-matrix fused interface where the cohesion and tensile strength were inferior (Figure 5). Thus, the angles between the overlapped fiber layers in the materials largely influenced the tensile properties as well as the burst performance of the MSFRSs.
3.3. Structure Analysis 3.3.1. FT-IR Spectroscopy
Figure 4. Burst test curves and comparisons of MSFRSs in the hydrated state. A-E represent the samples with overlapped fiber layers at angles of 08, 308, 608, 908 and plain SF membranes, respectively.
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The characteristic peaks of silk I structure around 1652 cm1 (Amide I), 1543 cm1 (Amide II), 1242 cm1 (Amide III) and 669 cm1 (Amide V), and silk II peaks around 1626 cm1 (Amide I), 1532 cm1 (Amide II), 1236 cm1 (Amide III) and 696 cm1 (Amide V) were reported in the literature.[28] As shown in the FT-IR spectra given in Figure 6,
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Figure 5. Photos of samples (A) and burst cracks (B) of overlapped fiber layers with different angles (a-d): 08, 308, 608 and 908, respectively.
the peaks at 1652 cm1, 1525 cm1 and 1239 cm1 indicated the presence of the a-helix, b-sheet and random coil structure in the samples of plain SF membrane and samples with multi-angle overlapping fiber layers at 08 and 608.[29] No significant difference in peak positions and stretches were found among these samples (Figure 6). The a-helical structure was mainly found in MSFRSs after treatment with
SDS; only a small portion of b-sheet structure was present, whereas the MSFRSs without SDS treatment possessed a mixture of a-helix and b-sheet structure due to the presence of the silk II structure dominated by the silk fibers. The integration of silk fibers with overlapped angles did not influence the secondary structure of silk protein matrix utilized in the processing.
Figure 6. The FT-IR spectra of pure SF membranes and MSFRSs: A, B, C, D and E represent the sample without SDS treatment, plain SF membrane and samples with multi-angle overlapping fiber layers at 08, 608 and 908, respectively.
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3.3.2. Wide angle X-Ray Diffraction (WAXD) The main reflection peaks of silk I are located at 12.18, 19.88, 24.18, 28.58 and 33.38, while the peaks at 9.18, 18.88, 20.58are assigned to the silk b-sheet structure.[30–32] Figure 7 shows the change in intensity, height and width of reflection peaks at 12.18, 19.88 and 24.18 in the WAXD pattern of MSFRSs. The presence of silk b-sheet and a-helical structures (silk I and II structures) was evidenced. No additional peaks were observed during the process; only the intensities of peaks changed among the different groups. Consistent with the FT-IR observations above, the X-ray diffraction data confirmed that the presence of silk fibers in the membrane matrix did not significantly influence the secondary structure of the continuous phase silk protein used as the binder. Figure 7. WAXD pattern of plain SF membranes and MSFRSs with multi-angle overlapped fiber layers at 08, 608 and 908 (A, B, C and D, respectively).
3.4. Cell Compatibility 3.4.1. Cell Morphology and Viability with MSFRSs in the Culture Medium Figure 8 shows that the HS-865-SK cells attached to the plates and spread evenly when the MSFRSs were floating in
the medium. Their filopodia exhibited normal morphology, and their shape was spindle-like, indicating good adhesion. There was no significant difference in the number and morphology of cells among different groups, indicating the
Figure 8. Qualitative analysis for the proliferation of HS-865-SK cells co-cultured with plain SF membrane and MSFRSs for 36 h: A, B and C represent the samples with laminated fiber layers at 608, 908 and the plain SF membrane, respectively; D is the blank tissue culture plate (TCP) containing culture medium (control).
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presence of MSFRSs in the culture medium did not influence cell attachment and proliferation. HS-865-SK cells were cultured in the medium containing plain SF membrane and MSFRSs, and cell viability was examined after 72 h using MTT analysis. As shown in Figure 9, with the increase of culture time, cell viability in each testing group increased. The number of cells increased slowly within 48 h. After 72 h with the multi-angle laminates at 608, 908, plain SF membrane and the control group (tissue culture plate; TCP), the optical density (OD) value increased to 1.09, 1.02, 1.13 and 1.27, respectively, indicating cell viability; more cells also gradually covered the surface of the plate wells of the control group. Cell viability on the 608 and 908 MSFRSs groups were lower than the control group (TCP without materials) at the end of experiment, but the difference was not significant (p > 0.05). The reasons for these differences are not clear, although residual SDS present in the MSFRSs might have influenced cell viability. Either more biocompatible surfactants or more careful extraction to remove residual SDS will be required. 3.4.2. Cell Attachment and Proliferation on Top of MSFRSs The attachment and proliferation of HS-865-SK cells cultured directly on the plain SF membranes and MSFRSs for 3 d are
Figure 9. Cell viability (O.D. value at 570 nm) of HS-865-SK cells co-cultured with plain SF membrane and MSFRSs for 72 h: the samples used for testing were multi-angle overlapped fiber layers at 608, 908, plain SF membrane, and the pure medium on the blank TCP (Error bars stand for mean SD, n ¼3 means each experiment was repeated for three times at least).
shown in Figure 10. The results showed that the cells attached to the fiber surfaces by discrete filopodia and microvilli, and the cells tend to grow along the fiber orientation. The cell growth on the plain SF and control
Figure 10. Qualitative analysis for HS-865-SK cells after attachment and proliferation for 3 d on the plain SF membrane and MSFRSs: A, B and C represent the sample with multi-angle overlapped fiber layers at 608, 908 and plain SF membrane, respectively; D is the blank TCP set as control group.
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group (TCP) was better than that on the MSFRSs groups in terms of cell morphology and viability. The reason might be that the overlapped fibers exposed to the membrane surface, forming a relatively rough surface that hindered cells attachment, although this has to be further proved. Furthermore, the growth of HS-865-SK cells on MSFRSs might be restricted by the exposed silk fibers due to steric hindrance. The observations obtained from this study will be helpful in fine-tuning the cell-material interactions and thus improving the cell compatibility of the MSFRSs in the future.
4. Conclusion Structural mimetic scaffolds reinforced with multi-angle silk fibers were successfully developed as laminate materials. Both the tensile properties and the burst performance were significantly improved by the overlapped fiber layers with defined angles (308 and 608) in the MSFRSs when compared to the plain silk scaffolds as well as those with 08 and 908 overlapped fiber layers. SDS pretreatment of the silk fibers further enhanced the effects, likely due to modification of fiber surfaces and thus interfacial cohesion between the fibers and material matrix. The presence of degummed silk fibers with different angles did not significantly impact the secondary structure and crystallization of the continuous phase silk used to bind the reinforced scaffold layers together. Cell attachment, morphological and viability studies also demonstrated that these new structural mimetic scaffolds were cell compatible. Acknowledgements: G.L. and J.L. contributed equally to this manuscript. This work was funded by the National Natural Science Foundation of China (NSFC) with project code 51273138 and State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University with project code LK1421. We would like to thank the support of Science and Technology Plans with project code SYN201403 and Science and Technology Program of Guangzhou with project code 2014J4100237.
Received: November 18, 2014; Revised: January 17, 2015; Published online:DOI: 10.1002/mabi.201400502 Keywords: fiber-reinforced; mechanical; multi-angle; scaffolds; silk
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Structural Mimetic Silk Fiber-Reinforced Composite Scaffolds Using Multi-Angle Fibers Gang Li, Jian Liu, Zhaozhu Zheng, Xiaoqin Wang,* David L. Kaplan* The fabrication of structural mimetic scaffolds reinforced with multi-angle silk fibers is described. Degummed silk fibers with a parallel arrangement of the fibers in a planar format were overlapped with successive layers organized at 08, 308, 608 and 908, respectively. The overlapped silk fiber layers were coated with silk solution (6 wt%) containing sodium dodecyl sulfate (SDS). The morphology, mechanical properties, structure and biocompatibility of the scaffolds were investigated. The mechanical properties of the scaffolds (tensile and burst) were characterized based on the angles of the fibers. Layers with an overlapping angle at 308 exhibited better mechanical performance (18 MPa) than the other groups. The results of Fourier Transform (FT) IR Spectroscopy (FT-IR) and X-ray Differentiation (XRD) analyses indicated that the presence of degummed silk fibers with different angles did not significantly impact secondary structure or crystallization of the fiber reinforced scaffolds. The attachment and growth of a human fibroblast cell line (HS-865-SK) on the reinforced scaffolds supported good cell compatibility. These new scaffolds have potential applications in tissue repairs where superior mechanical strength and cell compatibility are important.
In recent decades, fiber-reinforced composites (FRCs) have emerged as intriguing substitutes for plastics and metals in industrial applications[1] and represent successful biomaterials for biomedical applications.[2] Unlike homogeneous and isotropic materials, FRCs provide the possibility of designing materials with enhanced and tailored mechanical properties by selecting the material constituents (basic
G. Li, J. Liu, Z. Zheng, X. Wang National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, P. R. China E-mail: [email protected] D. L. Kaplan Department of Biomedical Engineering, Tufts University, 4 Colby St., Room 153, Medford, MA 02155, USA E-mail: [email protected]
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
matrix and reinforcing components, such as fibers) and setting their volume ratios.[1] Mechanical properties for structural engineering applications can be improved if the reinforcing fibers are properly blended with the matrix materials.[3–6] A critical goal for bioengineering functional and loadbearing tissues is the recapitulation of the robust mechanical properties intrinsic to the in vivo structures.[7] The use of FRCs in the preparation of medical materials and devices for soft and hard tissue replacement requires a wide selection range for the mechanical properties, as well as maintenance of biocompatibility.[8] Of the broad spectrum of biomimetic products, FRCs are often used for mechanically robust scaffolds consisting of a polymer matrix imbedded with high-strength fibers,[2,9] as well as biocompatible fibers, including silk fibers.[10] Compared to other fibers, silks consist of proteins produced naturally by silkworms and spiders, and are known as biocompatible, biodegradable, and extraordinarily robust biomaterials that are frequently
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1. Introduction
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utilized in biomaterial composites.[11–15] To produce robust matrices, a variety of functional inorganic nanostructures have been integrated with silk materials.[5,11,16,17] Yuan et al. reported a silk composite in which uniaxially-aligned and continuous-typed Bombyx mori silk fibers were embedded in a matrix of silk fibroin via a solution casting process. Compared to the individual material components, the overall mechanical properties as well as the thermal stability of the silk composites were significantly improved.[18,19] A typical application of composite materials where robust mechanics is required is for meniscus repair. Previous studies have used a variety of biodegradable scaffolds with or without the incorporation of stem cells, but the results have not been sufficient for clinical applications.[20] In order to develop mechanically robust scaffolds for meniscus repair, understanding and mimicking the complex internal architecture and function of the meniscus are critical.[21] The human meniscus is composed of an extracellular matrix where the organization and architecture of collagen fibers and proteoglycans are complex, conferring tissue-specific biomechanical characteristics. A longitudinal section through the meniscus shows that collagen fibers are arranged in three zones; superficial, lamellar and deep. The orientations of collagen fibers are of three types; circumferential, radial and random. Circumferential fibers are predominant and occupy the deep zone. Radial and random fibers are found in the lamellar zone and random fibers are scattered in the superficial zone.[22] In the present study, structural mimetic scaffolds reinforced with multi-angle silk fibers were fabricated and characterized in terms of mechanical strength, structure and cell interactions. The results indicated that silk fibers superimposed at defined angles (308 and 608) in the scaffolds significantly enhanced mechanical strength when compared to the scaffolds alone or the scaffolds with silk fibers superimposed at 08 and 908 angles. The fiber reinforcement technique and mechanisms elucidated in
this study can also be expanded to other mechanical strength-demanding tissue regeneration needs.
2. Experimental Section 2.1. Materials Degummed silk fibers were purchased from Xiehe Silk Corporation (Zhejiang, China). Lithium bromide was supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glycerol was provided by Shanghai Reagent Factory. Human dermal fibroblasts (Hs 865.Sk cells) were obtained from ATCC (American Type Culture Collection, Maryland, USA). Cell culture media were purchased from Wisent Corporation (Montreal, Canada). Other reagents were analytical grade.
2.2. Preparation of Multi-Angle Silk Fiber Reinforced Scaffolds (MSFRSs) The silk fibers were degummed by boiling in 0.02 m Na2CO3 for 30 min to remove sericin contaminants, as reported previously.[20] The degummed silk fibers (25%, w/v) were dissolved in 9.3 m LiBr solution at 60 8C for 4 h.[20] The mixing solution was dialyzed against pure water for 36 h to remove the lithium bromide. The solution was centrifuged at 12 000 g for 20 min at 4 8C to remove the insoluble fibrous debris and silk aggregates. Silk fibroin solution was then blended with 30 wt% glycerol prior to material processing to increase tenacity and surface smoothness of the regenerated silk fibroin scaffolds.[23] Double-sided tape was adhered onto the surface of a roller (diameter: 15 mm, length: 60 mm) and silk fibers were uniformly wound on the roller to form a single layer at a speed of 7 r min1. Along the axial direction, the double-sided tape was cut into two halves and removed from the roller, so that a fiber layer with parallel fiber arrangements was produced. Subsequently, the fiber layers were overlapped with different angles 08, 308, 608 and 908 respectively (Figure 1). The overlapped fiber layers were treated with 10% (w/v) sodium dodecyl sulfate (SDS) to increase cohesion between the fiber surfaces and the continuous silk fibroin matrix to improve mechanical performance. These laminates were then soaked in 1.5N mL of silk fibroin solution (6%, w/v), where N
Figure 1. Diagram of sampling approach (a) and different organizations of the fiber layers (b-e): 08, 308, 608 and 908, respectively.
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represents the number of layers of silk fibers with parallel arrangement. The samples were dried in the fume hood for 6 h, and subjected to the formation of a second layer of fibers. The process was repeated to obtain the laminates with different orientations of the constituent layers.
2.3. Characterization of MSFRSs 2.3.1. Morphology In order to compare the surface morphology of the MSFRSs before and after being treated with SDS, the samples were examined using a Scanning Electron Microscopy (SEM) (Hitachi S-4800, Tokyo, Japan). All specimens were sprayed with gold prior to imaging and observed at a voltage of 3 kV.
2.3.2. Mechanical Properties Tensile properties (samples shape and size: rectangle, 5010 mm) were examined in the hydrated state in three orientations (Figure 2a) using a load testing instrument (Model, Instron-5565, Norwood, MA) according to China standard GB/T 1040-2006. The testing conditions used included: tensile gap 28 mm; speed 20 mm min1 until rupture (T ¼ 25 8C, relative humidity (RH): 65%). The maximum strain and stress at rupture were observed and the Young’s modulus was calculated according to the slope of the stress-strain curve within the elastic region.[24,25] The burst properties of the samples (samples shape and size: round, diameter 100 2 mm) were examined for the composite materials in the hydrated state using a load testing instrument (Model: Instron3366, Norwood, MA). The measurement conditions: burst speed 20 mm min1 until rupture. The displacement-load curves were obtained for samples immersed in phosphate-buffered saline (PBS; pH ¼ 7.4) for 24 h before measurement. The measurements were repeated five times for each sample under standard testing environment (T ¼ 25 8C, RH ¼ 65%). Representative images are presented.
2.3.3. Structural Analysis The secondary structure of the MSFRSs was examined using Fourier Transform (FT) IR Spectroscopy with a MIRacleTM attenuated total reflection (ATR) Ge crystal cell in reflection mode. FT-IR wave
numbers were set from 4000 to 400 cm1 during 32 scans, with 2 cm1 resolution (Nicolet 5700, Thermo Electron Corp, Waltham, MA). The major vibration bands were attributed to specific chemical groups. The internal crystalline structure of the MSFRSs was examined via an X-ray diffractometer (X’Pert-Pro MPD, PANalytical BV, Almelo, Netherlands) using a CuKa radiation source. Two dimensional X-ray diffraction patterns were obtained at the following irradiation settings: Cu, Ka, wavelength 0.154 nm, range 58–608, 40 kV and 35 mA at a scanning rate of 58 min1.
2.4. Cell Culture and Seeding HS-865-SK cells were placed in 6-well plates at a density of 1.2 106 cells per well with 2.5 mL medium and maintained in high glucose Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Wisent Corporation, Canada) and 1% antibiotic/antimycotic at 37.0 0.2 8C in a humidified air incubator containing 5% CO2. The number of cells was counted using Cellometer Auto T4 (Nexcelom Bioscience LLC, USA). The scaffolds samples with 608 and 908 silk fiber compositions were selected and immersed in deionized water for 24 h before cell culture. Then a round-shaped sample (diameter: 15 mm, thickness: 0.15 mm) was cut from the scaffolds and placed on the bottom of 6well plates. Prior to seeding with cells, the samples were sterilized under 60Co radiation at 18 kGy (Irradiation Technology Institute of Soochow University, China). A total of 1.2 106 cells per well with 2.5 mL medium was added into the wells and incubated with the materials for 7 d.
2.5. Compatibility of MSFRSs 2.5.1. Cell Attachment and Proliferation Cells were cultured in the plate wells in which silk scaffolds (608 and 908 silk fibers) were either floated in the medium or fixed to the bottom of the wells. Plain silk membranes that were prepared in the absence of silk fibers served as controls. Cell attachment and proliferation at different time points were investigated. At each time point, three parallel samples in a testing group were taken out and subjected to measurements.
Figure 2. SEM images of fracture cross sectional views of MSFRSs, (a) MSFRS without treatment with SDS, (b) MSFRS treated with SDS.
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2.5.2. Cell Morphology Cell morphology and proliferation of each laminate group were observed using an Olympus phase contrast microscope TH4 200 (Osaka, Japan).
2.5.3. Cell Viability
surface hydrophilicity of the silk fibers and avoided bubbles formation during the blending process, thus providing higher contacting surface areas between the fibers and the continuous silk phase.[26,27]
3.2. Mechanical Properties
Methylthiazol tetrazolium (MTT) assay was conducted to determine cell viability in the presence of silk fiber-reinforced scaffolds. For viability, the cells were harvested, trypsinized and washed at 6, 24, 48 and 72 h intervals after seeding and then re-suspended gently in 1 mL PBS consisting of 10 mL of 12 mm MTT and 100 ml of SDS-HCl solution (fresh and filtered) for 12 h at 25 8C for staining. Absorbance was measured at 570 nm using a microplate reader (Model Synergy 2, Bio-Tek, Vermont, USA). All samples were run in triplicate.
2.6. Statistical Analysis Each experiment was repeated three times and all data sets are expressed in terms of mean and standard deviations. Statistical analysis was performed using one-way analysis of variance (ANOVA). The statistical difference between two groups of data was considered to be significant when P < 0.05.
3. Results and Discussion 3.1. Morphology of the MSFRSs Treated with SDS The morphologies of the MSFRSs treated with SDS before and after tensile measurement (method described above) are shown in Figure 2a. The cross-view of the fracture section during tensile measurement of the material without SDS treatment was rough and uneven, and moreover, part of the fibers pulled out of the sample. The results indicated a lack of cohesion between the fiber surface and the continuous silk fibroin matrix, which weakened mechanical performance. SDS is a commonly used surface active agent having both hydrophilic and hydrophobic groups, and has been used to improve the surface hydrophilicity of textiles due to its capabilities of emulsification, dispersion and solubilization.[23] In order to improve the mechanical properties of MSFRSs, prior to blending with SF solution, the silk fiber layers were treated by 10% (w/v) SDS for 0.5 h. The cross section view of the fracture surface of the material after the SDS treatment showed improved integration with few silk fibers being pulled out (Figure 2b). Thus, pre-treatment of the silk fibers with SDS facilitated material cohesion, giving rise to improved mechanical properties when compared to the untreated materials. The stress values of 608 sample before (and after) the treatment under axial, inclined and horizontal directions were 8.2 1.15 (9.7 1.65), 7.1 0.95 (10.2 1.05) and 7.2 1.02 (10.5 1.01) MPa, respectively. The reason is that the SDS treatment presumably increased
3.2.1. Tensile Tests The tensile stress and strain of the MSFRSs in the hydrated state were studied under axial, inclined and horizontal directions, respectively. As shown in Figure 3, the scaffolds with 08 overlapped fiber laminates exhibited a tensile stress above 40 MPa when the samples were measured under axial direction, much higher than other samples being tested (5–15 MPa). However, these samples showed the lowest isotropic tensile stress among all the samples tested. For example, the tensile stress in the inclined direction was only above 0.3 MPa, indicating that there was almost no tensile strength in the horizontal direction. This is mainly because there were few single fibers along the axial direction bearing continuous load. In contrast, the tensile stress of 308 and 608 overlapped samples under three directions was isotropic (10– 20 MPa). The 908 overlapped sample possessed high axial and horizontal tensile strength but low inclined tensile strength (3.3 MPa). In this case, the tensile behaviors were similar to that of the 08 overlapped sample, i.e., few continuous fibers were directly held by the two clamping ends on the instrument, and the fibers in the horizontal direction of the sample did not bear the tensile force. The plain SF membrane showed relatively high tensile strain (approximately 100%) due to its flexibility, but low tensile stress (3 MPa), and there was no significant difference between the strength values measured in all directions. The data suggested that embedding silk fibers in SF solution enhanced the strength of the SF laminate materials and the angle with regard to the fiber layers overlapped to each other had significant impact on the mechanical properties of the SF laminates. 3.2.2. Burst Tests The burst strength of various MSFRSs in the hydrated state was studied. As shown in Figure 4, the scaffolds with 308 overlapped fiber layers showed the highest burst strength above 160 N. For example, the burst strength of 08, 608 and 908 overlapped samples and the plain SF membranes were 15, 100, 75 and 20 N, respectively. When the material was subjected to burst pressure, it stretched in different directions and the region showing the lowest deformation strength was prone to crack first. Due to the uneven structural composition, the 08 overlapped samples showed the lowest tensile properties (see above) and burst strength
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Figure 3. The tensile curves and comparison of hydrated MSFRSs, a, b and c are tensile curves and stress comparisons of the samples under axial, inclined and horizontal directions, respectively. d is the results of a-c; A-E represent samples with multi-angle overlapping fiber layers at 08, 308, 608, 908 and the plain SF membrane, respectively.
under inclined and horizontal directions and linear cracks were observed (Figure 5B(a)). Therefore, these materials were prone to crack at the fiber-matrix fused interface where the cohesion and tensile strength were inferior (Figure 5). Thus, the angles between the overlapped fiber layers in the materials largely influenced the tensile properties as well as the burst performance of the MSFRSs.
3.3. Structure Analysis 3.3.1. FT-IR Spectroscopy
Figure 4. Burst test curves and comparisons of MSFRSs in the hydrated state. A-E represent the samples with overlapped fiber layers at angles of 08, 308, 608, 908 and plain SF membranes, respectively.
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The characteristic peaks of silk I structure around 1652 cm1 (Amide I), 1543 cm1 (Amide II), 1242 cm1 (Amide III) and 669 cm1 (Amide V), and silk II peaks around 1626 cm1 (Amide I), 1532 cm1 (Amide II), 1236 cm1 (Amide III) and 696 cm1 (Amide V) were reported in the literature.[28] As shown in the FT-IR spectra given in Figure 6,
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Figure 5. Photos of samples (A) and burst cracks (B) of overlapped fiber layers with different angles (a-d): 08, 308, 608 and 908, respectively.
the peaks at 1652 cm1, 1525 cm1 and 1239 cm1 indicated the presence of the a-helix, b-sheet and random coil structure in the samples of plain SF membrane and samples with multi-angle overlapping fiber layers at 08 and 608.[29] No significant difference in peak positions and stretches were found among these samples (Figure 6). The a-helical structure was mainly found in MSFRSs after treatment with
SDS; only a small portion of b-sheet structure was present, whereas the MSFRSs without SDS treatment possessed a mixture of a-helix and b-sheet structure due to the presence of the silk II structure dominated by the silk fibers. The integration of silk fibers with overlapped angles did not influence the secondary structure of silk protein matrix utilized in the processing.
Figure 6. The FT-IR spectra of pure SF membranes and MSFRSs: A, B, C, D and E represent the sample without SDS treatment, plain SF membrane and samples with multi-angle overlapping fiber layers at 08, 608 and 908, respectively.
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3.3.2. Wide angle X-Ray Diffraction (WAXD) The main reflection peaks of silk I are located at 12.18, 19.88, 24.18, 28.58 and 33.38, while the peaks at 9.18, 18.88, 20.58are assigned to the silk b-sheet structure.[30–32] Figure 7 shows the change in intensity, height and width of reflection peaks at 12.18, 19.88 and 24.18 in the WAXD pattern of MSFRSs. The presence of silk b-sheet and a-helical structures (silk I and II structures) was evidenced. No additional peaks were observed during the process; only the intensities of peaks changed among the different groups. Consistent with the FT-IR observations above, the X-ray diffraction data confirmed that the presence of silk fibers in the membrane matrix did not significantly influence the secondary structure of the continuous phase silk protein used as the binder. Figure 7. WAXD pattern of plain SF membranes and MSFRSs with multi-angle overlapped fiber layers at 08, 608 and 908 (A, B, C and D, respectively).
3.4. Cell Compatibility 3.4.1. Cell Morphology and Viability with MSFRSs in the Culture Medium Figure 8 shows that the HS-865-SK cells attached to the plates and spread evenly when the MSFRSs were floating in
the medium. Their filopodia exhibited normal morphology, and their shape was spindle-like, indicating good adhesion. There was no significant difference in the number and morphology of cells among different groups, indicating the
Figure 8. Qualitative analysis for the proliferation of HS-865-SK cells co-cultured with plain SF membrane and MSFRSs for 36 h: A, B and C represent the samples with laminated fiber layers at 608, 908 and the plain SF membrane, respectively; D is the blank tissue culture plate (TCP) containing culture medium (control).
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presence of MSFRSs in the culture medium did not influence cell attachment and proliferation. HS-865-SK cells were cultured in the medium containing plain SF membrane and MSFRSs, and cell viability was examined after 72 h using MTT analysis. As shown in Figure 9, with the increase of culture time, cell viability in each testing group increased. The number of cells increased slowly within 48 h. After 72 h with the multi-angle laminates at 608, 908, plain SF membrane and the control group (tissue culture plate; TCP), the optical density (OD) value increased to 1.09, 1.02, 1.13 and 1.27, respectively, indicating cell viability; more cells also gradually covered the surface of the plate wells of the control group. Cell viability on the 608 and 908 MSFRSs groups were lower than the control group (TCP without materials) at the end of experiment, but the difference was not significant (p > 0.05). The reasons for these differences are not clear, although residual SDS present in the MSFRSs might have influenced cell viability. Either more biocompatible surfactants or more careful extraction to remove residual SDS will be required. 3.4.2. Cell Attachment and Proliferation on Top of MSFRSs The attachment and proliferation of HS-865-SK cells cultured directly on the plain SF membranes and MSFRSs for 3 d are
Figure 9. Cell viability (O.D. value at 570 nm) of HS-865-SK cells co-cultured with plain SF membrane and MSFRSs for 72 h: the samples used for testing were multi-angle overlapped fiber layers at 608, 908, plain SF membrane, and the pure medium on the blank TCP (Error bars stand for mean SD, n ¼3 means each experiment was repeated for three times at least).
shown in Figure 10. The results showed that the cells attached to the fiber surfaces by discrete filopodia and microvilli, and the cells tend to grow along the fiber orientation. The cell growth on the plain SF and control
Figure 10. Qualitative analysis for HS-865-SK cells after attachment and proliferation for 3 d on the plain SF membrane and MSFRSs: A, B and C represent the sample with multi-angle overlapped fiber layers at 608, 908 and plain SF membrane, respectively; D is the blank TCP set as control group.
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group (TCP) was better than that on the MSFRSs groups in terms of cell morphology and viability. The reason might be that the overlapped fibers exposed to the membrane surface, forming a relatively rough surface that hindered cells attachment, although this has to be further proved. Furthermore, the growth of HS-865-SK cells on MSFRSs might be restricted by the exposed silk fibers due to steric hindrance. The observations obtained from this study will be helpful in fine-tuning the cell-material interactions and thus improving the cell compatibility of the MSFRSs in the future.
4. Conclusion Structural mimetic scaffolds reinforced with multi-angle silk fibers were successfully developed as laminate materials. Both the tensile properties and the burst performance were significantly improved by the overlapped fiber layers with defined angles (308 and 608) in the MSFRSs when compared to the plain silk scaffolds as well as those with 08 and 908 overlapped fiber layers. SDS pretreatment of the silk fibers further enhanced the effects, likely due to modification of fiber surfaces and thus interfacial cohesion between the fibers and material matrix. The presence of degummed silk fibers with different angles did not significantly impact the secondary structure and crystallization of the continuous phase silk used to bind the reinforced scaffold layers together. Cell attachment, morphological and viability studies also demonstrated that these new structural mimetic scaffolds were cell compatible. Acknowledgements: G.L. and J.L. contributed equally to this manuscript. This work was funded by the National Natural Science Foundation of China (NSFC) with project code 51273138 and State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University with project code LK1421. We would like to thank the support of Science and Technology Plans with project code SYN201403 and Science and Technology Program of Guangzhou with project code 2014J4100237.
Received: November 18, 2014; Revised: January 17, 2015; Published online:DOI: 10.1002/mabi.201400502 Keywords: fiber-reinforced; mechanical; multi-angle; scaffolds; silk
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