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Coaxial electrospun aligned tussah silk fibroin nanostructured fiber scaffolds embedded with hydroxyapatite–tussah silk fibroin nanoparticles for bone tissue engineering
Materials Science and Engineering C 58 (2016) 342–351
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Coaxial electrospun aligned tussah silk fibroin nanostructured fiber scaffolds embedded with hydroxyapatite–tussah silk fibroin nanoparticles for bone tissue engineering Weili Shao a, Jianxin He b,c,⁎, Feng Sang d, Bin Ding b,c, Li Chen a,⁎⁎, Shizhong Cui b,c, Kejing Li b,c, Qiming Han b,c, Weilin Tan b,c a
Key Laboratory of Advanced Textile Composites, Ministry of Education, Institute of Textile Composites, Tianjin Polytechnic University, Tianjin 300387, China College of Textiles, Zhongyuan University of Technology, Zhengzhou 450007, China c Collaborative Innovation Center of Textile and Garment Industry, Henan Province, Zhengzhou 450007, China d Department of Acquired Immune Deficiency Syndrome Treatment and Research Center, The First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou 450007, China b
a r t i c l e
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Article history: Received 20 May 2015 Received in revised form 23 July 2015 Accepted 25 August 2015 Available online 1 September 2015 Keywords: Tussah silk fibroin Hydroxyapatite Coaxial electrospinning Bone tissue engineering
a b s t r a c t The bone is a composite of inorganic and organic materials and possesses a complex hierarchical architecture consisting of mineralized fibrils formed by collagen molecules and coated with oriented hydroxyapatite. To regenerate bone tissue, it is necessary to provide a scaffold that mimics the architecture of the extracellular matrix in native bone. Here, we describe one such scaffold, a nanostructured composite with a core made of a composite of hydroxyapatite and tussah silk fibroin. The core is encased in a shell of tussah silk fibroin. The composite fibers were fabricated by coaxial electrospinning using green water solvent and were characterized using different techniques. In comparison to nanofibers of pure tussah silk, composite notably improved mechanical properties, with 90-fold and 2-fold higher initial modulus and breaking stress, respectively, obtained. Osteoblast-like MG-63 cells were cultivated on the composite to assess its suitability as a scaffold for bone tissue engineering. We found that the fiber scaffold supported cell adhesion and proliferation and functionally promoted alkaline phosphatase and mineral deposition relevant for biomineralization. In addition, the composite were more biocompatible than pure tussah silk fibroin or cover slip. Thus, the nanostructured composite has excellent biomimetic and mechanical properties and is a potential biocompatible scaffold for bone tissue engineering. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Natural bone forms according to a hierarchical architecture, components of which span nanometer to macroscopic scales. It is produced by natural biomineralization, in which organic templates control the growth of the inorganic phase [1]. The fundamental subunits of bone are mineralized collagen fibrils that consist of self-assembled triple helices of collagen molecules [2–4]. Hydroxyapatite nanocrystals grow on these fibrils with the crystallographic c-axis aligned with the axis of the fibril [5]. One strategy to repair or replace bone is to engineer bone tissue through a combination of scaffolds, implanted cells, and biologically active molecules [6–7]. An ideal bone scaffold should be biocompatible, biodegradable, and mechanically robust. This scaffold should facilitate early mineralization and support formation of new bone, while
⁎ Correspondence to: J.X. He, P.O. Box 110, College of Textiles, Zhongyuan University of Technology, 41 Zhongyuan Road, Zhengzhou, Henan Province 450007, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (J. He), [email protected] (L. Chen).
http://dx.doi.org/10.1016/j.msec.2015.08.046 0928-4931/© 2015 Elsevier B.V. All rights reserved.
simultaneously allowing replacement of old tissue [8–9]. In other words, the scaffold should have physical architecture and chemical composition similar to that of natural bone. Silk fibroin has gained significant interest as a scaffold due to excellent biocompatibility and biodegradability. Indeed, various strategies, including solvent casting, freeze drying, and salt leaching, have been developed to create three-dimensional silk scaffolds with high porosity and osteoconductivity. Ceramic components such as hydroxyapatite have been incorporated into these scaffolds to mimic the extracellular matrix [10–12]. To further enhance cell-scaffold interaction, nanofibers of silk fibroin have been used as alternatives to three-dimensional structures, because they more closely resemble the natural extracellular matrix and are also highly porous and interconnected [13–15]. Several approaches, including blending electrospinning and biomimetic mineralization, have been developed to fabricate such silk fibroin nanofibers, usually as a composite with hydroxyapatite, to better imitate natural bone [16–17]. For instance, Kim et al. [18] fabricated composite nanofibers using blending spinning and alternate soaking. Notably, the composite has better mechanical properties than similar scaffolds built from pure silk fibroin. Incorporation of hydroxyapatite into these composites further enhances specific biological activities
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Fig. 1. Schematic diagram of the set-up of coaxial electrospinning.
Fig. 2. SEM images (a–d) and diameter distribution (e) of nanostructured fibers with different core:shell mass ratios. (a) pure TSF; (b) 1:2; (c) 1:1; (d) 2:1.
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like cell adhesion, proliferation, differentiation, and mineralization [18–19]. Nevertheless, although incorporation of hydroxyapatite improves mechanical properties and biocompatibility, scaffolds that possess bone-like architecture have yet to be fabricated. Panda et al. [17] reported that the nature of the silk greatly affects the quality of the scaffold. Indeed, scaffolds built from eri (Philosamia ricini) or tasar (Antheraea mylitta) silk promote bone regeneration more effectively, and are more hydrophilic and mechanically stronger than those fabricated with silk fibroin from Bombyx mori [17]. Another silk with potential as scaffold is derived from tussah (Antheraea pernyi). Interestingly, this fibroin has higher alanine content than domestic silk. Thus, the crystalline region in tussah silk fibroin (TSF) mainly contains Ala repeat sequences instead of Gly-Ala-Gly-Ser repeats, as in domestic silk. Furthermore, TSF contains an Arg-Gly-Asp motif, which may function as a biological recognition signal, promote cell adhesion, and consequently render this protein suitable for biomedical applications [20–23]. Indeed, Altman et al. [24] and Minoura et al. [25] found that fibroblasts adhere with greater avidity to TSF than to silk from B. mori. In this study, we describe a new scaffold that is based on TSF and resembles parallel collagen fibers coated with hydroxyapatite crystals, similar to the architecture of native bone. The scaffold is fabricated as nanostructured composite with a core of nanoparticles encased in a shell of tussah silk. The nanoparticles are a composite of hydroxyapatite and TSF, and the composite is assembled by coaxial electrospinning. The morphology, structure, and mechanical properties of the scaffold were investigated. Its ability to promote bone formation was determined in vitro in terms of cell adhesion and proliferation, expression of alkaline phosphatase, and mineral deposition.
2. Materials and methods 2.1. Materials Tussah cocoons were obtained from Nanyang City in Henan Province, China. The human osteosarcoma cell line MG-63 and Dulbecco's modified Eagle's medium (DMEM) were purchased from the National Centre for Cell Science, Beijing, China. Trypsin-EDTA, penicillin–streptomycin, Triton X, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), the alkaline phosphatase assay kit, and the osteocalcin assay kit were obtained from Sigma-Aldrich (USA). Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were procured from Gibco (Singapore). Materials and reagents were used as received without further purification. 2.2. Preparation of aqueous tussah silk fibroin Cocoons were degummed thrice at 98 °C for 30 min using 0.5 wt.% Na2CO3. Degummed tussah fibers were dissolved by diluting 35-fold in 9 M aqueous lithium thiocyanate, and then stirring at 55 °C for 1 h. Subsequently, the solution was filtered to remove the remaining particulates, and dialyzed for 3 days against distilled water. A 31 wt.% aqueous solution was prepared by dialysis in 35 wt.% PEG according to published methods [26–27]. 2.3. Preparation of hydroxyapatite–TSF composite nanoparticles A solution of Ca(OH)2 and H3PO4 was prepared to a final Ca:P molar ratio of 1.67. This solution was added dropwise into 10 wt.% aqueous
Fig. 3. (a) High magnification TEM images of composite nanoparticles; the inset shows polycrystalline diffraction ring; (b) TEM images of composite nanoparticles; TEM images of composite with different core:shell mass ratios. (e) 1:2; (f) 1:1; (g) 2:1.
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Fig. 4. (a) TG curves, (b) FTIR spectra, and (c) XRD patterns of pure TSF nanofibers, composite fibers with different core:shell mass ratios, composite particles and pure hydroxyapatite particles.
tussah fibroin with ammonia to adjust the pH to ~10–10.5. The concentration of aqueous TSF was selected to achieve a final concentration of 5 wt.% composite particles. The mixture was stirred for 2 h at 60 °C with ultrasonication and then aged for 48 h at room temperature. Particles were separated from the liquor by filtration, washed 3–4 times with distilled water. Nanoparticles were resuspended in 0.4 wt.% citric acid to a final concentration of 31 wt.%. Finally, the pH of the slurry was adjusted to ~10–10.5 by using ammonia.
2.4. Fabrication The nanostructured fibers were fabricated using a syringe-like apparatus in which a needle is coaxially placed within another needle (Fig. 1). The internal and external needles were connected by silicone tubing to syringes containing core and shell feed solutions, respectively. The core solution was 31 wt.% composite nanoparticles, while the shell solution was 31 wt.% purified TSF. Electrospinning was performed at an applied voltage of 20 kV, solution-dispensing rate of ~0.1–0.3 mL/h, and ambient conditions of ~ 45–50% humidity and ~ 25–28 °C. Nanofibers were collected with a roller at a distance of 18 cm from the needle tip. Nanofibers with core:shell mass ratios of 1:2, 1:1, and 2:1 were obtained by manipulating the spinning flow of individual needles.
Fig. 5. Tensile curves of aligned composite fiber mats with different core:shell mass ratio and non-woven pure TSF nanofiber mats.
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Table 1 Mechanical properties of pure TSF nanofiber and composite nanostructured fiber mats. Samples
Tensile strength (MPa)
Elongation at break (%)
Young's modulus (MPa)
Pure TSF (non-woven) Pure TSF 1:2 1:1 2:1
1.7 ± 0.2 2.7 ± 0.5 3.0 ± 0.2 5.3 ± 0.6 3.1 ± 0.4
90.5 ± 16.2 101.9 ± 5.1 80.1 ± 4.5 62.6 ± 5.2 4.9 ± 1.1
0.4 ± 0.1 0.8 ± 0.1 27.5 ± 3.2 70.2 ± 4.3 69.4 ± 6.2
filtered CuKα radiation at 0.1542 nm. The operating voltage and current were 40 kV and 30 mA, respectively. Tensile mechanical properties were determined at 20 °C and 65% relative humidity using an Instron tester (Model 3365, USA). For this analysis, we prepared nanostructured fiber mats with a width of 10 mm, initial length of 30 mm, and thickness of 0.15–0.30 mm. Tests were performed with a gauge length of 15 mm, rate of 10 mm/min, and applied load of 10 N. The tensile strength and elongation data reported here are the median of ten tests. 2.6. Seeding and proliferation
2.5. Characterization Electrospun nanostructured fibers were coated with gold and examined using a Hitachi TM-1000 scanning electron microscope (SEM) operating at 15 kV. Average fiber diameter was determined from at least 100 fibers based on SEM images. Structure was further characterized using a Hitachi H-800 transmission electron microscope (TEM) operating at 100 kV. For TEM, composite fibers were spotted on carbon-coated copper grids. Samples were also analyzed by thermogravimetry using the Perkin Elmer TGA. Samples were heated to 1000 °C at 10.0 °C/min. FTIR spectra were collected with a Nicolet Nexus 670 FTIR spectrometer using a sample of 1 mg powdered fibers dispersed in 300 mg KBr. A total of 100 scans were recorded at a resolution of 2 cm− 1 . X-ray diffraction data were collected at a scanning speed of 0.02°/s using a Rigaku-D/Max-2550 PC diffractometer (Japan) emitting Ni-
Human osteosarcoma MG-63 cells were maintained at subconfluent density in DMEM supplemented with 10% v/v FBS. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2, and media were refreshed twice a week. Fibers were sterilized by immersing in 70 wt.% ethanol, washing twice with sterile PBS. MG-63 cells were seeded on sterile scaffolds at 5 × 104 cells/mL/well in a 24-well plate. Media were replenished every 48 h. For comparison, cells were also seeded on cover slips. Cell adhesion and proliferation, alkaline phosphatase activity, and osteocalcin production were measured at 1, 4, and 7 days after seeding, respectively. 2.7. MTT assay Cell proliferation was monitored with an MTT assay. Briefly, cells were washed with PBS and incubated for 4 h with 200 μg MTT.
Fig. 6. Proliferation and morphology observation of MG-63 cells cultured onto composite, pure TSF nanofibers and cover slip for up to 7 days. (a) Histogram of cell proliferation by MTT assay; (b) SEM micrographs. All values are expressed as mean ± SD from four different replicates. *Statistical significance of p b 0.05. SD, standard deviation.
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Subsequently, 400 μL DMSO was added to dissolve the purple MTT formazan crystals that were formed. After the suspension was subjected to shaking for 15 min on an oscillator, the absorbance of the supernatant was measured at 570 nm using a microplate reader. 2.8. Morphology of cells growing on nanostructured fibers Non-adherent cells were washed away using PBS. Cells attached to nanostructured fibers were fixed for 3 h at room temperature with 4 wt.% paraformaldehyde. Samples were then dehydrated using a
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graded ethanol series, dried under vacuum, sputter-coated with gold, and examined by SEM. Elemental analysis of mineral deposits was performed using energy-dispersive spectrometry. Fluorescence staining was also performed to assess cytoskeletal structure in adherent cells. Briefly, cell-coated scaffolds were fixed with 4.0 wt.% paraformaldehyde and washed with PBS. Cells were permeabilized with 1 mL 0.1% v/v Triton X-100 and washed thrice with PBS. Subsequently, samples were incubated for 30 min at room temperature in the dark with 150 μL rhodamine-phalloidin (Sigma) diluted 1:40 in PBS. Subsequently, samples were stained for 15 min at
Fig. 7. (a) Fluorescent microscope images of MG-63 cells seeded on composite, pure TSF nanofibers and cover slip after 1, 4 and 7 days of culture. F-actin filaments (red) and nucleus (blue) were stained by rhodamine-phalloidin and DAPI, respectively. Quantitative analysis of cell proliferation and adherence to different substrates: (b) fluorescent intensity of nuclear; (c) fluorescent intensity of cytoplasm; (d) nuclear ratio index; and (e) cell area (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.).
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37 °C with 0.8 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) in PBS. Then, samples were examined with a Nikon Eclipse TE 2000-U fluorescent microscope (AVON, MA).
2.9. Alkaline phosphatase activity Alkaline phosphatase activity was measured by p-nitrophenol release. Briefly, samples were incubated at room temperature for 30 min with 400 μL p-nitrophenyl phosphate (Phosphatase Substrate Kit; Pierce Biotechnology, Rockford, IL). The reaction was terminated with 200 μL 2 M NaOH and absorbance was measured at 405 nm.
2.10. Osteocalcin expression Osteocalcin content was determined by ELISA. Briefly, 80 μL culture supernatant was collected, clarified by centrifugation, and stored in an EP pipe. From the clarified supernatant, 10 μL was added to ELISA plates coated with polylysine, which were then incubated for 30 min at 37 °C, and washed five times in a microplate washer (Bio-Tek EL404). Subsequently, wells were treated for 30 min at 37 °C with 50 μL enzyme labeling reagent, and washed as described above. A reagent dye dispenser was added prior to incubation at 37 °C in the dark. The reaction was terminated by addition of 50 μL termination solution and osteocalcin content was determined from absorbance at 450 nm.
Fig. 8. (a) ALP activity of MG-63 cells seeded on composite, pure TSF nanofibers and cover slip after 1, 4, 7 and 10 days of culture. (b) OC produced by MG-63 cells cultured on composite, pure TSF nanofibers and cover slip after 1, 4 and 7 days of culture. SEM images of the mineral deposition produced by MG-63 cells on aligned pure TSF nanofibers (c, e) and composite (d, f) after 4 and 7 days of culture. (g), (h) EDS spectra analysis for the detection of mineral deposits after culturing MG-63 cells for 7 days with the two fiber scaffolds. All values are expressed as mean ± SD from three independent measurements. *Statistical significance of p b 0.05. SD, standard deviation.
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2.11. Statistical analysis Data were collected in quadruplicate (n = 4) and reported as mean ± standard deviation where indicated. Statistical analysis was performed using t-test, and p values less than 0.05 were considered significant.
3. Results and discussion
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The same absorption bands were observed in composite, confirming that composite nanoparticles were incorporated. X-ray diffraction curves (Fig. 4c) of TSF nanofibers included two wide peaks at 2θ 12.06° and 23.50°, which are characteristic of αhelical silk fibroin. X-ray diffraction curves of composite displayed a similar diffraction curve to TSF nanofibers. However, three weaker diffraction peaks at 2θ 25.90°, 31.80°, and 49.20° were observed, which correspond to the (002), (211), and (222) crystal planes, respectively, of hydroxyapatite. Thus, X-ray diffraction data further verified that hydroxyapatite is embedded in composite.
3.1. Morphology and structure 3.2. Mechanical properties Aqueous tussah silk and a composite of tussah silk and hydroxyapatite were used to fabricate the shell and core, respectively, of bone-like nanofibers. To avoid toxicity from organic solvents, green water solvent was used during fabrication. SEM images of electrospinning composite fibers and pure silk nanofibers are shown in Fig. 2, and indicate that almost all fibers were highly parallel. Further, the composite nanoparticles in the core were encased well in the TSF shell, especially at a core:shell mass ratio of 1:2. However, beading was observed in composite fibers with a mass ratio of 2:1, indicating aggregation of composite nanoparticles in the core. SEM images were also used to estimate the diameter of the fiber and its distribution (Fig. 2e). Aqueous TSF showed good spinnability, and slightly flat fibers with a diameter of 370–590 nm were obtained. Incorporation of a composite core visibly increased the diameter of the resulting fiber. For instance, the diameter of composite fibers with a 1:1 ratio of tussah silk and composite nanoparticles was between 510 nm and 840 nm, almost twice the diameter of silk nanofibers. However, the core:shell ratio did not significantly affect fiber diameter, which increased only marginally from 860 nm to 950 nm as the ratio increased from 1:2 to 2:1. The use of composite nanoparticles containing TSF in core was motivated by the need to improve miscibility between TSF in the shell and hydroxyapatite in the core. High magnification TEM images show incorporation of nanoscale hydroxyapatite sub-crystallites without uniform crystallographic orientation (Fig. 3a). The structure of composite nanoparticles was further investigated by selected area electron diffraction. Based on this analysis, two diffraction arcs could be ascribed to the (002) and (112) planes of hydroxyapatite (Fig. 3a, inset), with corresponding interplanar spacing of 0.3435 nm and 0.2790 nm, respectively (Fig. 3a). The nanocrystals were needle-shaped with a length of 98.7 nm and width of 3.2 nm (Fig. 3b). TEM images of composite are shown in Fig. 3c–e. The composite fibers fabricated with a core-shell design are distinctive, and a fuzzy boundary between the core and shell implies good miscibility. As the core:shell mass ratio increased from 1:2 to 1:1, the ratio between their thickness was observed to increase from 0.32 to 0.45. However, composite with a 2:1 ratio showed an obvious aggregation of those nanoparticles, which could not be encased by the TSF shell. Analysis by thermogravimetry indicated that composite nanoparticles in the core are 94.5 wt.% hydroxyapatite and 5.5 wt.% TSF (Fig. 4a). Similar measurements for the core:shell mass ratio in composite indicated that the core comprised 39.2 wt.%, 52.6 wt.%, and 69.5 wt.% of the weight, while the shell comprised the remaining 60.8 wt.%, 47.4 wt.%, and 30.5 wt.%. These values are in good agreement with the designed weight ratios of 1:2, 1:1, and 2:1, respectively. Fig. 4b shows FTIR spectra collected from composite, pure TSF nanofibers, and composite nanoparticles. Nanofibers of pure TSF exhibited a total of three bands at 1650 cm−1 (amide I), 1541 cm−1 (amide II), and 1259 cm−1 (amide III), which could be attributed to α-helices or random coils. Composite nanoparticles showed prominent peaks at 1638 cm−1 (amide I) and 1541 cm−1 (amide II), suggesting the presence of silk fibroin in β-sheets. Additionally, these nanoparticles exhibited absorption bands at 1037 cm− 1 and 962 cm−1, which could be group in hydroxyapatite. assigned to stretching vibration of the PO3− 4
Ample mechanical strength is a property essential for a tissue scaffold. Tensile curves of composite sub-microfiber mats with varying core:shell ratios are shown in Fig. 5. Non-woven nanofiber mats of pure TSF were soft and flexible and had low initial modulus and high elongation. Fiber orientation was observed to significantly affect tensile properties. The breaking stress of a non-woven mat was 1.7 MPa, while that of an aligned mat was 2.7 MPa (Table 1). The tensile properties of composite were determined to a significant extent by the amount of composite nanoparticles in the core. The materials gradually changed in mechanical behavior from soft to rigid with increase in the amount of composite nanoparticles. This trend is also reflected in the obvious increase in Young's modulus. For instance, the Young's modulus of a composite with a 1:1 core:shell ratio was 90fold higher than that of nanofibers of pure silk, while the breaking stress was 2-fold higher. In contrast, breaking strain decreased from 101.9% to 62.6%. However, composites with a 2:1 ratio were fragile and broke before yield. Stress and strain at the breaking point decreased to 3.1 MP and 4.9%, respectively, possibly due to high inorganic content and aggregation in the core. 3.3. Cell proliferation Bone scaffolds for implantation should be biocompatible, should facilitate cell proliferation, and should not elicit an immune response, leading to rejection. Therefore, osteoblast-like MG-63 cells were seeded on silk and composite to evaluate biocompatibility. The MTT assay did not show significant differences in cell proliferation after 1 day (p N 0.05; Fig. 6a), although nanofibers of tussah silk elicited slightly better proliferation than cover slip between day 4 and day 7. However, cell proliferation in sub-microfiber composites was significantly higher after 4 days (p b 0.05), indicating that nanoparticles in the core enhance cell proliferation. The morphology of MG-63 cells adhering to fiber scaffolds was examined by SEM. It is clear from Fig. 6b that cells attached to and proliferated along the fiber axis. Beginning at 4 days, cells were observed to attach with higher density to composite scaffolds than to silk nanofibers or cover slip. Meanwhile, some cells appeared a matured spindle-like shape with bi-polar or tri-polar extensions on composite scaffolds. However, an extensive network of lamellipodia and filopodia was woven and integrated into composite scaffolds after 7 days. In contrast, cells proliferated without cohesion on the other two substrates. Cells were also stained to further examine morphology (Fig. 7a). Cells seeded on fibers acquired the shape of an extended spindle after 1 day, and grew at similar density. In contrast, those seeded on cover slip were irregular, polygonal, or triangular in shape and were without filopodia. After 4 days, the composite scaffold was more densely coated with cells than the two other substrates; the difference in cell density became remarkable after 7 days. Finally, cells grew in only one direction along fibers, a consequence of highly parallel architecture. Several parameters including nuclear fluorescent intensity (NFI), cytoplasmic fluorescent intensity (CFI), and cell area were measured (Fig. 7b-d). Except on day 1, NFI was markedly higher in cells cultured on composite scaffolds than on other substrates. CFI followed a similar
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trend, although the difference was less marked. Because NFI and CFI reflect cell density, it is clear that composite scaffolds contained the highest number of cells after day 1. NCI, the ratio between NFI and CFI, is a quantitative measure of cell proliferation (Fig. 7d). Based on this ratio, composite elicited cell proliferation most effectively. Notably, the NCI on pure silk was even lower than on cover slip at day 4, composite nanoparticles, not silk, enhance cell proliferation, presumably by promoting interaction between cells and the extracellular matrix. Similar results were obtained when the cell area was calculated in order to compare cell attachment (Fig. 7e). Thus, all data indicate that composite scaffolds are highly biocompatible and promote cell adhesion and proliferation.
Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (Nos. 51203196, U1204510), and the financial supports from the Program for Science &Technology Innovation Talents in Universities of Henan Province of China (No. 15HASTIT024), Zhengzhou Science and Technology Program (141PPTGG400, 131PLJRC653) and the Key Science and Technology Research Program of Education Department of Henan, Province of China (14A540003; 14A540006) are also gratefully acknowledged.
References 3.4. Biomineralization Alkaline phosphatase is a key component of bone matrix vesicles and catalyzes the formation of calcium apatites in the extracellular matrix [28–34]. Therefore, the enzyme is a marker of bone maturation, and activity was measured in cells seeded on different scaffolds (Fig. 8a). Enzyme activity was similarly low at day 1 (p N 0.05) on all three substrates. However, activity was significantly enhanced after day 4 in cells growing on composite, peaking on day 7. Indeed, enzyme activity on day 7 was found to be 162% and 211% higher in composite scaffolds than in pure TSF or cover slip, respectively. Notably, alkaline phosphatase activity markedly decreased at day 10 in cells growing on composite material, suggesting that bone development has reached advanced stages [35]. In contrast, activity continued to increase slowly in cells growing on pure TSF or cover slip, reflecting slower development. Osteocalcin is another marker of bone development, and its expression tended to follow a similar trajectory as alkaline phosphatase (Fig. 8b) [36]. Thus, osteocalcin production was significantly higher after 4 days in composites than in pure TSF or cover slip. Nevertheless, production on cover slip and TSF was found to increase slowly. These results provide further evidence that composite nanoparticles encased in TSF provide a suitable biomimetic environment to elicit cell proliferation and differentiation. Matrix mineralization is a measure of the success of bone formation in vitro [37]. Fig. 8c–f shows SEM images of mineral deposits after 4 and 7 days in cells growing on pure TSF and composite scaffolds. At 4 days, globular minerals were deposited in cells growing on composite, but not in those growing on pure TSF. Mineralization was observed to increase after 7 days, and particles were observed to coalesce on composite. However, mineral density in TSF scaffolds was still significantly lesser than that in composite materials. Elemental analysis by energy-dispersive spectrometry confirmed the presence of calcium and phosphorus in mineral deposits (Fig. 16g, h). The calcium:phosphorus ratio in minerals accumulating on TSF and composite was 1.42 ± 0.15 and 1.56 ± 0.17, respectively, at 7 days. These values are slightly lower than 1.67, the stoichiometric ratio in hydroxyapatite, suggesting calcium deficiency, a well-known condition in natural bone [38].
4. Conclusions Highly parallel composite nanostructured fibers, which imitate collagen fibrils in natural bone, were fabricated by coaxial electrospinning in green water solvent. These composite nanostructured fibers consisted of a composite of hydroxyapatite and tussah silk in a core encased in a shell of tussah silk, and had good mechanical properties. In addition, biological assays indicated that the composite elicited cell adhesion, proliferation, and bone formation more effectively than pure silk or cover slip. Thus, these composites represent a novel set of biomaterials with potential application as scaffolds in tissue engineering and bone regeneration.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Coaxial electrospun aligned tussah silk fibroin nanostructured fiber scaffolds embedded with hydroxyapatite–tussah silk fibroin nanoparticles for bone tissue engineering Weili Shao a, Jianxin He b,c,⁎, Feng Sang d, Bin Ding b,c, Li Chen a,⁎⁎, Shizhong Cui b,c, Kejing Li b,c, Qiming Han b,c, Weilin Tan b,c a
Key Laboratory of Advanced Textile Composites, Ministry of Education, Institute of Textile Composites, Tianjin Polytechnic University, Tianjin 300387, China College of Textiles, Zhongyuan University of Technology, Zhengzhou 450007, China c Collaborative Innovation Center of Textile and Garment Industry, Henan Province, Zhengzhou 450007, China d Department of Acquired Immune Deficiency Syndrome Treatment and Research Center, The First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou 450007, China b
a r t i c l e
i n f o
Article history: Received 20 May 2015 Received in revised form 23 July 2015 Accepted 25 August 2015 Available online 1 September 2015 Keywords: Tussah silk fibroin Hydroxyapatite Coaxial electrospinning Bone tissue engineering
a b s t r a c t The bone is a composite of inorganic and organic materials and possesses a complex hierarchical architecture consisting of mineralized fibrils formed by collagen molecules and coated with oriented hydroxyapatite. To regenerate bone tissue, it is necessary to provide a scaffold that mimics the architecture of the extracellular matrix in native bone. Here, we describe one such scaffold, a nanostructured composite with a core made of a composite of hydroxyapatite and tussah silk fibroin. The core is encased in a shell of tussah silk fibroin. The composite fibers were fabricated by coaxial electrospinning using green water solvent and were characterized using different techniques. In comparison to nanofibers of pure tussah silk, composite notably improved mechanical properties, with 90-fold and 2-fold higher initial modulus and breaking stress, respectively, obtained. Osteoblast-like MG-63 cells were cultivated on the composite to assess its suitability as a scaffold for bone tissue engineering. We found that the fiber scaffold supported cell adhesion and proliferation and functionally promoted alkaline phosphatase and mineral deposition relevant for biomineralization. In addition, the composite were more biocompatible than pure tussah silk fibroin or cover slip. Thus, the nanostructured composite has excellent biomimetic and mechanical properties and is a potential biocompatible scaffold for bone tissue engineering. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Natural bone forms according to a hierarchical architecture, components of which span nanometer to macroscopic scales. It is produced by natural biomineralization, in which organic templates control the growth of the inorganic phase [1]. The fundamental subunits of bone are mineralized collagen fibrils that consist of self-assembled triple helices of collagen molecules [2–4]. Hydroxyapatite nanocrystals grow on these fibrils with the crystallographic c-axis aligned with the axis of the fibril [5]. One strategy to repair or replace bone is to engineer bone tissue through a combination of scaffolds, implanted cells, and biologically active molecules [6–7]. An ideal bone scaffold should be biocompatible, biodegradable, and mechanically robust. This scaffold should facilitate early mineralization and support formation of new bone, while
⁎ Correspondence to: J.X. He, P.O. Box 110, College of Textiles, Zhongyuan University of Technology, 41 Zhongyuan Road, Zhengzhou, Henan Province 450007, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (J. He), [email protected] (L. Chen).
http://dx.doi.org/10.1016/j.msec.2015.08.046 0928-4931/© 2015 Elsevier B.V. All rights reserved.
simultaneously allowing replacement of old tissue [8–9]. In other words, the scaffold should have physical architecture and chemical composition similar to that of natural bone. Silk fibroin has gained significant interest as a scaffold due to excellent biocompatibility and biodegradability. Indeed, various strategies, including solvent casting, freeze drying, and salt leaching, have been developed to create three-dimensional silk scaffolds with high porosity and osteoconductivity. Ceramic components such as hydroxyapatite have been incorporated into these scaffolds to mimic the extracellular matrix [10–12]. To further enhance cell-scaffold interaction, nanofibers of silk fibroin have been used as alternatives to three-dimensional structures, because they more closely resemble the natural extracellular matrix and are also highly porous and interconnected [13–15]. Several approaches, including blending electrospinning and biomimetic mineralization, have been developed to fabricate such silk fibroin nanofibers, usually as a composite with hydroxyapatite, to better imitate natural bone [16–17]. For instance, Kim et al. [18] fabricated composite nanofibers using blending spinning and alternate soaking. Notably, the composite has better mechanical properties than similar scaffolds built from pure silk fibroin. Incorporation of hydroxyapatite into these composites further enhances specific biological activities
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Fig. 1. Schematic diagram of the set-up of coaxial electrospinning.
Fig. 2. SEM images (a–d) and diameter distribution (e) of nanostructured fibers with different core:shell mass ratios. (a) pure TSF; (b) 1:2; (c) 1:1; (d) 2:1.
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like cell adhesion, proliferation, differentiation, and mineralization [18–19]. Nevertheless, although incorporation of hydroxyapatite improves mechanical properties and biocompatibility, scaffolds that possess bone-like architecture have yet to be fabricated. Panda et al. [17] reported that the nature of the silk greatly affects the quality of the scaffold. Indeed, scaffolds built from eri (Philosamia ricini) or tasar (Antheraea mylitta) silk promote bone regeneration more effectively, and are more hydrophilic and mechanically stronger than those fabricated with silk fibroin from Bombyx mori [17]. Another silk with potential as scaffold is derived from tussah (Antheraea pernyi). Interestingly, this fibroin has higher alanine content than domestic silk. Thus, the crystalline region in tussah silk fibroin (TSF) mainly contains Ala repeat sequences instead of Gly-Ala-Gly-Ser repeats, as in domestic silk. Furthermore, TSF contains an Arg-Gly-Asp motif, which may function as a biological recognition signal, promote cell adhesion, and consequently render this protein suitable for biomedical applications [20–23]. Indeed, Altman et al. [24] and Minoura et al. [25] found that fibroblasts adhere with greater avidity to TSF than to silk from B. mori. In this study, we describe a new scaffold that is based on TSF and resembles parallel collagen fibers coated with hydroxyapatite crystals, similar to the architecture of native bone. The scaffold is fabricated as nanostructured composite with a core of nanoparticles encased in a shell of tussah silk. The nanoparticles are a composite of hydroxyapatite and TSF, and the composite is assembled by coaxial electrospinning. The morphology, structure, and mechanical properties of the scaffold were investigated. Its ability to promote bone formation was determined in vitro in terms of cell adhesion and proliferation, expression of alkaline phosphatase, and mineral deposition.
2. Materials and methods 2.1. Materials Tussah cocoons were obtained from Nanyang City in Henan Province, China. The human osteosarcoma cell line MG-63 and Dulbecco's modified Eagle's medium (DMEM) were purchased from the National Centre for Cell Science, Beijing, China. Trypsin-EDTA, penicillin–streptomycin, Triton X, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), the alkaline phosphatase assay kit, and the osteocalcin assay kit were obtained from Sigma-Aldrich (USA). Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were procured from Gibco (Singapore). Materials and reagents were used as received without further purification. 2.2. Preparation of aqueous tussah silk fibroin Cocoons were degummed thrice at 98 °C for 30 min using 0.5 wt.% Na2CO3. Degummed tussah fibers were dissolved by diluting 35-fold in 9 M aqueous lithium thiocyanate, and then stirring at 55 °C for 1 h. Subsequently, the solution was filtered to remove the remaining particulates, and dialyzed for 3 days against distilled water. A 31 wt.% aqueous solution was prepared by dialysis in 35 wt.% PEG according to published methods [26–27]. 2.3. Preparation of hydroxyapatite–TSF composite nanoparticles A solution of Ca(OH)2 and H3PO4 was prepared to a final Ca:P molar ratio of 1.67. This solution was added dropwise into 10 wt.% aqueous
Fig. 3. (a) High magnification TEM images of composite nanoparticles; the inset shows polycrystalline diffraction ring; (b) TEM images of composite nanoparticles; TEM images of composite with different core:shell mass ratios. (e) 1:2; (f) 1:1; (g) 2:1.
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Fig. 4. (a) TG curves, (b) FTIR spectra, and (c) XRD patterns of pure TSF nanofibers, composite fibers with different core:shell mass ratios, composite particles and pure hydroxyapatite particles.
tussah fibroin with ammonia to adjust the pH to ~10–10.5. The concentration of aqueous TSF was selected to achieve a final concentration of 5 wt.% composite particles. The mixture was stirred for 2 h at 60 °C with ultrasonication and then aged for 48 h at room temperature. Particles were separated from the liquor by filtration, washed 3–4 times with distilled water. Nanoparticles were resuspended in 0.4 wt.% citric acid to a final concentration of 31 wt.%. Finally, the pH of the slurry was adjusted to ~10–10.5 by using ammonia.
2.4. Fabrication The nanostructured fibers were fabricated using a syringe-like apparatus in which a needle is coaxially placed within another needle (Fig. 1). The internal and external needles were connected by silicone tubing to syringes containing core and shell feed solutions, respectively. The core solution was 31 wt.% composite nanoparticles, while the shell solution was 31 wt.% purified TSF. Electrospinning was performed at an applied voltage of 20 kV, solution-dispensing rate of ~0.1–0.3 mL/h, and ambient conditions of ~ 45–50% humidity and ~ 25–28 °C. Nanofibers were collected with a roller at a distance of 18 cm from the needle tip. Nanofibers with core:shell mass ratios of 1:2, 1:1, and 2:1 were obtained by manipulating the spinning flow of individual needles.
Fig. 5. Tensile curves of aligned composite fiber mats with different core:shell mass ratio and non-woven pure TSF nanofiber mats.
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Table 1 Mechanical properties of pure TSF nanofiber and composite nanostructured fiber mats. Samples
Tensile strength (MPa)
Elongation at break (%)
Young's modulus (MPa)
Pure TSF (non-woven) Pure TSF 1:2 1:1 2:1
1.7 ± 0.2 2.7 ± 0.5 3.0 ± 0.2 5.3 ± 0.6 3.1 ± 0.4
90.5 ± 16.2 101.9 ± 5.1 80.1 ± 4.5 62.6 ± 5.2 4.9 ± 1.1
0.4 ± 0.1 0.8 ± 0.1 27.5 ± 3.2 70.2 ± 4.3 69.4 ± 6.2
filtered CuKα radiation at 0.1542 nm. The operating voltage and current were 40 kV and 30 mA, respectively. Tensile mechanical properties were determined at 20 °C and 65% relative humidity using an Instron tester (Model 3365, USA). For this analysis, we prepared nanostructured fiber mats with a width of 10 mm, initial length of 30 mm, and thickness of 0.15–0.30 mm. Tests were performed with a gauge length of 15 mm, rate of 10 mm/min, and applied load of 10 N. The tensile strength and elongation data reported here are the median of ten tests. 2.6. Seeding and proliferation
2.5. Characterization Electrospun nanostructured fibers were coated with gold and examined using a Hitachi TM-1000 scanning electron microscope (SEM) operating at 15 kV. Average fiber diameter was determined from at least 100 fibers based on SEM images. Structure was further characterized using a Hitachi H-800 transmission electron microscope (TEM) operating at 100 kV. For TEM, composite fibers were spotted on carbon-coated copper grids. Samples were also analyzed by thermogravimetry using the Perkin Elmer TGA. Samples were heated to 1000 °C at 10.0 °C/min. FTIR spectra were collected with a Nicolet Nexus 670 FTIR spectrometer using a sample of 1 mg powdered fibers dispersed in 300 mg KBr. A total of 100 scans were recorded at a resolution of 2 cm− 1 . X-ray diffraction data were collected at a scanning speed of 0.02°/s using a Rigaku-D/Max-2550 PC diffractometer (Japan) emitting Ni-
Human osteosarcoma MG-63 cells were maintained at subconfluent density in DMEM supplemented with 10% v/v FBS. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2, and media were refreshed twice a week. Fibers were sterilized by immersing in 70 wt.% ethanol, washing twice with sterile PBS. MG-63 cells were seeded on sterile scaffolds at 5 × 104 cells/mL/well in a 24-well plate. Media were replenished every 48 h. For comparison, cells were also seeded on cover slips. Cell adhesion and proliferation, alkaline phosphatase activity, and osteocalcin production were measured at 1, 4, and 7 days after seeding, respectively. 2.7. MTT assay Cell proliferation was monitored with an MTT assay. Briefly, cells were washed with PBS and incubated for 4 h with 200 μg MTT.
Fig. 6. Proliferation and morphology observation of MG-63 cells cultured onto composite, pure TSF nanofibers and cover slip for up to 7 days. (a) Histogram of cell proliferation by MTT assay; (b) SEM micrographs. All values are expressed as mean ± SD from four different replicates. *Statistical significance of p b 0.05. SD, standard deviation.
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Subsequently, 400 μL DMSO was added to dissolve the purple MTT formazan crystals that were formed. After the suspension was subjected to shaking for 15 min on an oscillator, the absorbance of the supernatant was measured at 570 nm using a microplate reader. 2.8. Morphology of cells growing on nanostructured fibers Non-adherent cells were washed away using PBS. Cells attached to nanostructured fibers were fixed for 3 h at room temperature with 4 wt.% paraformaldehyde. Samples were then dehydrated using a
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graded ethanol series, dried under vacuum, sputter-coated with gold, and examined by SEM. Elemental analysis of mineral deposits was performed using energy-dispersive spectrometry. Fluorescence staining was also performed to assess cytoskeletal structure in adherent cells. Briefly, cell-coated scaffolds were fixed with 4.0 wt.% paraformaldehyde and washed with PBS. Cells were permeabilized with 1 mL 0.1% v/v Triton X-100 and washed thrice with PBS. Subsequently, samples were incubated for 30 min at room temperature in the dark with 150 μL rhodamine-phalloidin (Sigma) diluted 1:40 in PBS. Subsequently, samples were stained for 15 min at
Fig. 7. (a) Fluorescent microscope images of MG-63 cells seeded on composite, pure TSF nanofibers and cover slip after 1, 4 and 7 days of culture. F-actin filaments (red) and nucleus (blue) were stained by rhodamine-phalloidin and DAPI, respectively. Quantitative analysis of cell proliferation and adherence to different substrates: (b) fluorescent intensity of nuclear; (c) fluorescent intensity of cytoplasm; (d) nuclear ratio index; and (e) cell area (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.).
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37 °C with 0.8 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) in PBS. Then, samples were examined with a Nikon Eclipse TE 2000-U fluorescent microscope (AVON, MA).
2.9. Alkaline phosphatase activity Alkaline phosphatase activity was measured by p-nitrophenol release. Briefly, samples were incubated at room temperature for 30 min with 400 μL p-nitrophenyl phosphate (Phosphatase Substrate Kit; Pierce Biotechnology, Rockford, IL). The reaction was terminated with 200 μL 2 M NaOH and absorbance was measured at 405 nm.
2.10. Osteocalcin expression Osteocalcin content was determined by ELISA. Briefly, 80 μL culture supernatant was collected, clarified by centrifugation, and stored in an EP pipe. From the clarified supernatant, 10 μL was added to ELISA plates coated with polylysine, which were then incubated for 30 min at 37 °C, and washed five times in a microplate washer (Bio-Tek EL404). Subsequently, wells were treated for 30 min at 37 °C with 50 μL enzyme labeling reagent, and washed as described above. A reagent dye dispenser was added prior to incubation at 37 °C in the dark. The reaction was terminated by addition of 50 μL termination solution and osteocalcin content was determined from absorbance at 450 nm.
Fig. 8. (a) ALP activity of MG-63 cells seeded on composite, pure TSF nanofibers and cover slip after 1, 4, 7 and 10 days of culture. (b) OC produced by MG-63 cells cultured on composite, pure TSF nanofibers and cover slip after 1, 4 and 7 days of culture. SEM images of the mineral deposition produced by MG-63 cells on aligned pure TSF nanofibers (c, e) and composite (d, f) after 4 and 7 days of culture. (g), (h) EDS spectra analysis for the detection of mineral deposits after culturing MG-63 cells for 7 days with the two fiber scaffolds. All values are expressed as mean ± SD from three independent measurements. *Statistical significance of p b 0.05. SD, standard deviation.
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2.11. Statistical analysis Data were collected in quadruplicate (n = 4) and reported as mean ± standard deviation where indicated. Statistical analysis was performed using t-test, and p values less than 0.05 were considered significant.
3. Results and discussion
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The same absorption bands were observed in composite, confirming that composite nanoparticles were incorporated. X-ray diffraction curves (Fig. 4c) of TSF nanofibers included two wide peaks at 2θ 12.06° and 23.50°, which are characteristic of αhelical silk fibroin. X-ray diffraction curves of composite displayed a similar diffraction curve to TSF nanofibers. However, three weaker diffraction peaks at 2θ 25.90°, 31.80°, and 49.20° were observed, which correspond to the (002), (211), and (222) crystal planes, respectively, of hydroxyapatite. Thus, X-ray diffraction data further verified that hydroxyapatite is embedded in composite.
3.1. Morphology and structure 3.2. Mechanical properties Aqueous tussah silk and a composite of tussah silk and hydroxyapatite were used to fabricate the shell and core, respectively, of bone-like nanofibers. To avoid toxicity from organic solvents, green water solvent was used during fabrication. SEM images of electrospinning composite fibers and pure silk nanofibers are shown in Fig. 2, and indicate that almost all fibers were highly parallel. Further, the composite nanoparticles in the core were encased well in the TSF shell, especially at a core:shell mass ratio of 1:2. However, beading was observed in composite fibers with a mass ratio of 2:1, indicating aggregation of composite nanoparticles in the core. SEM images were also used to estimate the diameter of the fiber and its distribution (Fig. 2e). Aqueous TSF showed good spinnability, and slightly flat fibers with a diameter of 370–590 nm were obtained. Incorporation of a composite core visibly increased the diameter of the resulting fiber. For instance, the diameter of composite fibers with a 1:1 ratio of tussah silk and composite nanoparticles was between 510 nm and 840 nm, almost twice the diameter of silk nanofibers. However, the core:shell ratio did not significantly affect fiber diameter, which increased only marginally from 860 nm to 950 nm as the ratio increased from 1:2 to 2:1. The use of composite nanoparticles containing TSF in core was motivated by the need to improve miscibility between TSF in the shell and hydroxyapatite in the core. High magnification TEM images show incorporation of nanoscale hydroxyapatite sub-crystallites without uniform crystallographic orientation (Fig. 3a). The structure of composite nanoparticles was further investigated by selected area electron diffraction. Based on this analysis, two diffraction arcs could be ascribed to the (002) and (112) planes of hydroxyapatite (Fig. 3a, inset), with corresponding interplanar spacing of 0.3435 nm and 0.2790 nm, respectively (Fig. 3a). The nanocrystals were needle-shaped with a length of 98.7 nm and width of 3.2 nm (Fig. 3b). TEM images of composite are shown in Fig. 3c–e. The composite fibers fabricated with a core-shell design are distinctive, and a fuzzy boundary between the core and shell implies good miscibility. As the core:shell mass ratio increased from 1:2 to 1:1, the ratio between their thickness was observed to increase from 0.32 to 0.45. However, composite with a 2:1 ratio showed an obvious aggregation of those nanoparticles, which could not be encased by the TSF shell. Analysis by thermogravimetry indicated that composite nanoparticles in the core are 94.5 wt.% hydroxyapatite and 5.5 wt.% TSF (Fig. 4a). Similar measurements for the core:shell mass ratio in composite indicated that the core comprised 39.2 wt.%, 52.6 wt.%, and 69.5 wt.% of the weight, while the shell comprised the remaining 60.8 wt.%, 47.4 wt.%, and 30.5 wt.%. These values are in good agreement with the designed weight ratios of 1:2, 1:1, and 2:1, respectively. Fig. 4b shows FTIR spectra collected from composite, pure TSF nanofibers, and composite nanoparticles. Nanofibers of pure TSF exhibited a total of three bands at 1650 cm−1 (amide I), 1541 cm−1 (amide II), and 1259 cm−1 (amide III), which could be attributed to α-helices or random coils. Composite nanoparticles showed prominent peaks at 1638 cm−1 (amide I) and 1541 cm−1 (amide II), suggesting the presence of silk fibroin in β-sheets. Additionally, these nanoparticles exhibited absorption bands at 1037 cm− 1 and 962 cm−1, which could be group in hydroxyapatite. assigned to stretching vibration of the PO3− 4
Ample mechanical strength is a property essential for a tissue scaffold. Tensile curves of composite sub-microfiber mats with varying core:shell ratios are shown in Fig. 5. Non-woven nanofiber mats of pure TSF were soft and flexible and had low initial modulus and high elongation. Fiber orientation was observed to significantly affect tensile properties. The breaking stress of a non-woven mat was 1.7 MPa, while that of an aligned mat was 2.7 MPa (Table 1). The tensile properties of composite were determined to a significant extent by the amount of composite nanoparticles in the core. The materials gradually changed in mechanical behavior from soft to rigid with increase in the amount of composite nanoparticles. This trend is also reflected in the obvious increase in Young's modulus. For instance, the Young's modulus of a composite with a 1:1 core:shell ratio was 90fold higher than that of nanofibers of pure silk, while the breaking stress was 2-fold higher. In contrast, breaking strain decreased from 101.9% to 62.6%. However, composites with a 2:1 ratio were fragile and broke before yield. Stress and strain at the breaking point decreased to 3.1 MP and 4.9%, respectively, possibly due to high inorganic content and aggregation in the core. 3.3. Cell proliferation Bone scaffolds for implantation should be biocompatible, should facilitate cell proliferation, and should not elicit an immune response, leading to rejection. Therefore, osteoblast-like MG-63 cells were seeded on silk and composite to evaluate biocompatibility. The MTT assay did not show significant differences in cell proliferation after 1 day (p N 0.05; Fig. 6a), although nanofibers of tussah silk elicited slightly better proliferation than cover slip between day 4 and day 7. However, cell proliferation in sub-microfiber composites was significantly higher after 4 days (p b 0.05), indicating that nanoparticles in the core enhance cell proliferation. The morphology of MG-63 cells adhering to fiber scaffolds was examined by SEM. It is clear from Fig. 6b that cells attached to and proliferated along the fiber axis. Beginning at 4 days, cells were observed to attach with higher density to composite scaffolds than to silk nanofibers or cover slip. Meanwhile, some cells appeared a matured spindle-like shape with bi-polar or tri-polar extensions on composite scaffolds. However, an extensive network of lamellipodia and filopodia was woven and integrated into composite scaffolds after 7 days. In contrast, cells proliferated without cohesion on the other two substrates. Cells were also stained to further examine morphology (Fig. 7a). Cells seeded on fibers acquired the shape of an extended spindle after 1 day, and grew at similar density. In contrast, those seeded on cover slip were irregular, polygonal, or triangular in shape and were without filopodia. After 4 days, the composite scaffold was more densely coated with cells than the two other substrates; the difference in cell density became remarkable after 7 days. Finally, cells grew in only one direction along fibers, a consequence of highly parallel architecture. Several parameters including nuclear fluorescent intensity (NFI), cytoplasmic fluorescent intensity (CFI), and cell area were measured (Fig. 7b-d). Except on day 1, NFI was markedly higher in cells cultured on composite scaffolds than on other substrates. CFI followed a similar
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trend, although the difference was less marked. Because NFI and CFI reflect cell density, it is clear that composite scaffolds contained the highest number of cells after day 1. NCI, the ratio between NFI and CFI, is a quantitative measure of cell proliferation (Fig. 7d). Based on this ratio, composite elicited cell proliferation most effectively. Notably, the NCI on pure silk was even lower than on cover slip at day 4, composite nanoparticles, not silk, enhance cell proliferation, presumably by promoting interaction between cells and the extracellular matrix. Similar results were obtained when the cell area was calculated in order to compare cell attachment (Fig. 7e). Thus, all data indicate that composite scaffolds are highly biocompatible and promote cell adhesion and proliferation.
Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (Nos. 51203196, U1204510), and the financial supports from the Program for Science &Technology Innovation Talents in Universities of Henan Province of China (No. 15HASTIT024), Zhengzhou Science and Technology Program (141PPTGG400, 131PLJRC653) and the Key Science and Technology Research Program of Education Department of Henan, Province of China (14A540003; 14A540006) are also gratefully acknowledged.
References 3.4. Biomineralization Alkaline phosphatase is a key component of bone matrix vesicles and catalyzes the formation of calcium apatites in the extracellular matrix [28–34]. Therefore, the enzyme is a marker of bone maturation, and activity was measured in cells seeded on different scaffolds (Fig. 8a). Enzyme activity was similarly low at day 1 (p N 0.05) on all three substrates. However, activity was significantly enhanced after day 4 in cells growing on composite, peaking on day 7. Indeed, enzyme activity on day 7 was found to be 162% and 211% higher in composite scaffolds than in pure TSF or cover slip, respectively. Notably, alkaline phosphatase activity markedly decreased at day 10 in cells growing on composite material, suggesting that bone development has reached advanced stages [35]. In contrast, activity continued to increase slowly in cells growing on pure TSF or cover slip, reflecting slower development. Osteocalcin is another marker of bone development, and its expression tended to follow a similar trajectory as alkaline phosphatase (Fig. 8b) [36]. Thus, osteocalcin production was significantly higher after 4 days in composites than in pure TSF or cover slip. Nevertheless, production on cover slip and TSF was found to increase slowly. These results provide further evidence that composite nanoparticles encased in TSF provide a suitable biomimetic environment to elicit cell proliferation and differentiation. Matrix mineralization is a measure of the success of bone formation in vitro [37]. Fig. 8c–f shows SEM images of mineral deposits after 4 and 7 days in cells growing on pure TSF and composite scaffolds. At 4 days, globular minerals were deposited in cells growing on composite, but not in those growing on pure TSF. Mineralization was observed to increase after 7 days, and particles were observed to coalesce on composite. However, mineral density in TSF scaffolds was still significantly lesser than that in composite materials. Elemental analysis by energy-dispersive spectrometry confirmed the presence of calcium and phosphorus in mineral deposits (Fig. 16g, h). The calcium:phosphorus ratio in minerals accumulating on TSF and composite was 1.42 ± 0.15 and 1.56 ± 0.17, respectively, at 7 days. These values are slightly lower than 1.67, the stoichiometric ratio in hydroxyapatite, suggesting calcium deficiency, a well-known condition in natural bone [38].
4. Conclusions Highly parallel composite nanostructured fibers, which imitate collagen fibrils in natural bone, were fabricated by coaxial electrospinning in green water solvent. These composite nanostructured fibers consisted of a composite of hydroxyapatite and tussah silk in a core encased in a shell of tussah silk, and had good mechanical properties. In addition, biological assays indicated that the composite elicited cell adhesion, proliferation, and bone formation more effectively than pure silk or cover slip. Thus, these composites represent a novel set of biomaterials with potential application as scaffolds in tissue engineering and bone regeneration.
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