Green fabrication of porous silk fibroin/graphene oxide hybrid scaffolds for bone tissue engineering

Abstract

The fabrication of novel scaffolds providing mechanical support and promoting tissue regeneration is urgently needed in bone tissue engineering. In the present study, three-dimensional (3-D) porous scaffolds based on silk fibroin (SF) and graphene oxide (GO) were prepared by freeze-drying. It is also worth noting that glycerol was blended with the SF solution and induced silk crystallization during the lyophilization process, and such non-use of organic solvents realized the concept of “green” preparation. With the incorporation of GO, the average pore diameter of the scaffolds decreased and their porosity changes were slight. The glycerol-treated scaffolds showed a stable silk II structure and their compressive modulus were improved significantly after GO was added. The biological performance of the SF/GO scaffolds including biodegradation behavior, drug release properties and biocompatibility were also investigated. Compared with the SF scaffold, the incorporation of GO enhanced the ability of the scaffolds to resist enzyme degradation. Simvastatin (SIM) was chosen as a model drug for release studies from the scaffolds. The results indicated that SIM release was dependant on the GO content within the scaffolds. MC3T3-E1 cells cultured in the hybrid scaffolds with moderate GO content demonstrated obviously enhanced osteogenic proliferation compared to that in the SF scaffolds without or with high concentration GO. Furthermore, the incorporation of SIM was beneficial to the growth of osteoblasts and the SF/GO/SIM scaffolds showed better cytocompatibility. These new 3-D SF/GO hybrid scaffolds with sustained drug release capacity would offer promising potential as platforms for bone tissue regeneration.

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1. Introduction

Designing and creating a desirable scaffold serving as a temporary artificial extracellular matrix to support the regeneration of functional tissues is always a challenge in tissue engineering. An ideal scaffold should have some necessary features including interconnected pore structure, sufficient mechanical properties, excellent biocompatibility and suitable biodegradation rate. Furthermore, convenient incorporation of bioactive molecules such as drug or growth factor is an additional design feature needed for tissue substitutes.

Silk fibroin (SF) is a naturally occurring protein produced by the domestic silkworm, Bombyx mori.¹ During decades of research, SF has been recognized as a potentially useful biomaterial due to its excellent mechanical properties, impressive biocompatibility, minimal inflammatory and long-term biodegradability.^2–7 Depending on the various requirements of different applications, SF has been designed and prepared as films,⁸ scaffolds,⁹ hydrogels¹⁰ or non-woven mats¹¹ and all these SF-based materials show promising future for tissue engineering scaffolds used in skin,¹² bone,¹³ blood vessel,¹⁴ and nerve tissue¹⁵ regeneration.

Recently, graphene oxide (GO), a novel single atomic monolayer prepared from natural graphite,¹⁶ has attracted considerable attention due to its unique structure,¹⁷ outstanding mechanical property¹⁸ and high thermal and electrical conductivity.^19,20 As one of the most promising two-dimensional (2-D) materials, GO has been employed in various applications including nanocomposite materials,²¹ nanoelectronic devices,²² and transparent conductors.²³ Additionally, studies have revealed the feasibility of GO as a potential candidate for biomedical applications owing to its good biocompatibility.^24–32 For example, GO or functionalized GO has been proven to be an effective drug delivery system;^24–26 graphene and its chemical derivatives could support cellular adhesion, proliferation and even induce the differentiation of stem cells.^27,28 Furthermore, low concentrations of GO has been certified to be nontoxic and it can be recognized as a safe nanomaterial in vitro and even in vivo.²⁹ Most importantly, the incorporation of GO into scaffolds has been reported to enhance the osteogenic differentiation of Mesenchymal stem cells (MSCs),^30–32 indicating the potential of the GO-incorporated scaffolds used for bone tissue engineering.

Simvastatin (SIM), an inhibitor of the competitive 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase, has been safely administered orally for over 20 years.³³ It has also been proven that the effects of SIM on bone formation are associated with an increase in the expression of bone morphogenetic protein-2 (BMP-2) mRNA and enhanced the vascular endothelial growth factor (VEGF) expression, an important factor in osteoblast differentiation.^34–36 Consequently, SIM is clinically recognized as a kind of promising drug for bone disease treatment.

To satisfy the various requirements of a scaffold for bone regeneration, we fabricate a novel three-dimensional (3-D) SF/GO hybrid scaffold loaded with SIM. Before our work, the SF/GO based materials had been studied by several researchers. The strong composite film was prepared by facile solution casting of SF/GO hydrogels.³⁷ The novel ultrathin SF/GO membranes with remarkable mechanical properties were fabricated by the layer-by-layer technique.³⁸ Recently, a novel, flexible and biocompatible 2-D SF/GO composite film was successfully prepared in our laboratory.³⁹ Unfortunately, the drug-loaded 3-D scaffolds based on SF/GO have not caused widespread concern yet. Many researchers ignore the possibilities that the incorporation of GO would enable the single component SF based materials more applicable as a specific scaffold for bone tissue regeneration. Additionally, glycerol was firstly used as a plasticizer in the SF/GO scaffold where glycerol regulated protein crystallization to achieve water stability instead of the common methanol treatment or water annealing, which would avoid the use of organic solvents during the fabrication process. Compared with the previous studies, the present study is aimed at designing and preparing a hybrid scaffold with interconnected pores, desirable biodegradability and excellent biocompatibility using a simple and green method. Furthermore, SIM was used as a model drug to evaluate the influences of GO on the drug release behavior. Also, the impacts of GO content in the hybrid scaffolds and SIM release from the porous scaffolds on the cell behaviour were investigated in vitro. Therefore, the SF/GO hybrid scaffold with satisfactory physical properties and sustained drug release performance can be employed as a scaffolding option for bone tissue regeneration.

2. Experimental section

2.1 Materials

Bombyx mori raw silk fibers were purchased from Dingsheng Silk Co. Ltd (Wujiang, China). Graphene oxide was purchased from XFNano Materials Tech Co. Ltd (Nanjing, China). Simvastatin was purchased from TCI (Shanghai, China). Murine osteoblastic cells MC3T3-E1 were purchased from TongPai Biological Tech Co. Ltd (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen. Cell Tracker CM-DiI (Molecular Probes, Invitrogen) was purchased from Wegene Biological Tech Co. Ltd (Shanghai, China). Glycerol (G-2025), collagenase-IA and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich Trading Co. Ltd (Shanghai, China).

2.2 Preparation of the SF/GO hybrid scaffolds and SF/GO/SIM scaffolds

SF solution was produced as previously described.¹ Briefly, the Bombyx mori silk fibers were degummed in a 0.05 wt% Na₂CO₃ solution at 100 °C, rinsed thoroughly with distilled water and dried at 60 °C for one night. The extracted silk fibers were dissolved in ternary solvent of CaCl₂ : CH₃CH₂OH : H₂O (1 : 2 : 8 molar ratio) at 72 °C for 1 hour. Then, a ∼3.5 wt% SF solution was obtained after dialysis in distilled water for 4 days. GO powder was dispersed in distilled water by ultrasonication for 30 minutes, adjusting the GO concentration for 2 mg ml⁻¹. The pH value of the GO suspension was modulated to 10 by sodium hydroxide solution (1 mol l⁻¹). Then the GO suspension was added into the diluted SF solution (2 wt%) drop wise, and a homogeneous solution was obtained after stirring for 30 minutes. The final GO contents in the hybrid scaffolds were 0.1, 0.2, 0.5 and 1.0 wt% against to the weight of SF. Glycerol was used as the cross-linking agent to obtain the insoluble scaffolds. To investigate the appropriate dosage of glycerol, SF/GO hybrid solution was mixed with glycerol at SF/glycerol weight ratios of 100/10, 100/20, 100/30 and 100/40, respectively. The mixed solution was poured into stainless steel dish, frozen at −40 °C for 2 h, followed by lyophilization for 48 h to form the SF/GO hybrid scaffolds.

The SF/GO scaffolds incorporated with SIM were prepared by the method similar to that of the SF/GO scaffolds. Firstly, SIM powder was dispersed in distilled water by ultrasonication to form 10 mg ml⁻¹ dispersion. The uniform SIM dispersion was added into the above-mentioned SF/GO/glycerol solution (SIM/SF w/w ratios of 1 : 40) with constant stirring. Finally, after freezing for 2 h, the homogeneous mixture was lyophilization for 48 h to form the SF/GO/SIM scaffolds.

2.3 Characterizations

2.3.1 Water solubility of the SF/GO scaffold blended with different glycerol content. SF/GO 0.5 wt% scaffold was chosen to evaluate the influence of glycerol content on the water solubility performance. The samples (cylinder of 10 mm in diameter and 8 mm in height) were immersed in 50 ml distilled water and kept at 37 °C for 0.5 h, 2 h, 4 h, 6 h, 8 h and 10 h. At each time point, the scaffolds were removed from the water, dried at 40 °C and weighted. The residual mass (%) was obtained by dividing the residual weight by the initial weight of the scaffold. Three replicates were performed for each appointed time.

2.3.2 Morphology and pore structure of the SF/GO scaffolds. The cross-section morphology of the SF/GO porous scaffolds was sputtered coated with gold and observed using a field-emission scanning electron microscope (SEM, JEOL, Japan). The pore sizes were determined by averaging random 50 pores from the SEM images. The porosity of the scaffolds was measured by liquid displacement, using hexane as the displacement liquid.⁹ A pre-weighted dry sample was immersed in a defined volume (V₁) of hexane in a graduated cylinder for 10 min. The total volume of the hexane and the hexane-impregnated scaffold was recorded as V₂. Then the hexane-impregnated scaffold was removed from the cylinder and the residual hexane volume was recorded as V₃. The porosity of the scaffold was expressed as: porosity (%) = (V₁ − V₃)/(V₂ − V₃).

2.3.3 Structure analysis and mechanical properties of the SF/GO scaffolds. The structure of the various scaffolds were analyzed by X-ray diffraction (XRD) on Miniflex II (Rigaku, Japan) diffractometer with Cu Kα radiation (λ = 1.54178 Å). All samples were cut into micro-particles before analysis and measured with 2θ ranging from 5° to 40°. The Fourier transform infrared (FT-IR) was also preformed on a Nicolet Magna-IR 750 spectrometer in the range of 4000–500 cm⁻¹. DSC was measured by a NETZSCH DSC 200F3 instrument at a heating rate of 10 °C min⁻¹ under a dry nitrogen gas flow of 50 ml min⁻¹. The compressive properties of the scaffolds (cylinder of 16 mm in diameter and 10 mm in height) were measured with a loading velocity of 5 mm min⁻¹ at 20 °C on an electronics universal tensile testing machine (TRAPEZIUM, Japan) with a 500 N loading cell. The load was eliminated when the sample was compressed to 60% of its original height. The elastic modulus was determined as the slope of the linear-elastic region of the strength–strain curve. For each sample, 5 measurements were carried out and the average values were reported.

2.3.4 Biodegradation of the SF/GO scaffolds. The SF/GO scaffolds were incubated at 37 °C in 50 ml of phosphate-buffered saline (PBS) containing 1 U ml⁻¹ collagenase-IA for 28 days. Groups of sample residuals were carefully rinsed in distilled water, collected at each appointed time and weighted. The degradation solution was replenished with fresh enzyme every 2 days. The morphologic changes of the various scaffolds after degradation were examined by SEM (JEOL, Japan).

2.3.5 In vitro release of SIM from the SF/GO scaffolds. The release of SIM from the SF/GO scaffolds was investigated by incubating the drug-loaded samples in 50 ml PBS at 37 °C for up to 30 days. At pre-determined time points, 3 ml of the solution was removed and immediately replaced with an equal volume of fresh media to keep the volume constant. The amount of SIM released was quantified by UV-spectroscopy at 238.5 nm and the concentration was calculated using a linear standard curve. All experiments were performed in triplicate.

2.3.6 Cell viability assay. Murine osteoblastic cells MC3T3-E1 were used to evaluate the potential of the SF/GO and SF/GO/SIM scaffolds served as options for bone tissue engineering. The content of GO in the drug-loaded scaffolds was 0.5 wt% against to the weight of SF. Viability of cells was assessed by laser scanning confocal microscopy (LSCM, Leica company, Germany), SEM and MTT assays.

The various scaffolds with a diameter of 1 cm were placed in the 24-well tissue culture plates (Corning Inc., USA), sterilized by γ-ray irradiation and rinsed with sterilized PBS prior to cell seeding. MC3T3-E1 cells at a density of 1 × 10⁵ cells per ml were seeded onto the hybrid scaffolds in the 24-well plates. Then the cell-seeded scaffolds were incubated in DMEM/F12 medium with 10% FBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The medium in each well was replaced every 2 days.

To investigate the distribution of MC3T3-E1 cells in the hybrid scaffolds, the cells were labeled by CM-DiI dye (2 μg ml⁻¹). Then the labeled cells were seeded in the porous scaffolds and observed by LSCM after incubation for 5 and 10 days. The samples for SEM analysis were fixed with 2.5% glutaraldehyde at 4 °C for one night, followed by 3 washes in sterilized PBS, and dehydrated with gradient ethanol solutions. Specimens were then sputter coated with gold in vacuum and examined using a JEOL JSM-7001F SEM. MTT tests were performed on day 1, 4, 7, and 10 after seeding in vitro. Briefly, the cell-seeded-scaffolds were incubated in MTT solution (5 mg ml⁻¹ in PBS) at 37 °C for 4 h, and then the blue formazan crystals produced by viable cells were dissolved in DMSO for 10 minutes. The optical density (OD) of formazan was measured on a Synergy HT (BIO-TEK) microplate reader at 490 nm. Three MTT assay replicates were performed for each sample at each appointed time.

2.3.7 Statistical analysis. Statistical comparisons were performed using SPSS 16.0 statistical software, and differences at P < 0.05 were considered statistically significant.

3. Results and discussion

3.1 Water solubility

The water solubility of scaffolds with different glycerol content at different time points was provided in Fig. 1. SF/GO scaffold without glycerol rapidly dissolved in water within several minutes, while the samples with 10 wt% and 20 wt% glycerol also had large solubility in ten hours which resulted in the poor water stability. When the glycerol content increased to 30 wt% and 40 wt%, the water stability of the scaffolds was significantly improved, which implied that the stable silk I and silk II crystal structure was formed after glycerol was incorporated. Water stability is critically important for a scaffold due to its further application in vitro and in vivo. So the water-stable SF/GO scaffold with 30 wt% glycerol was used in the present study and they were rinsed with distilled water for 24 h to remove the residual glycerol and dried at ambient temperature for the other tests.

Fig. 1 Water solubility of the SF/GO 0.5 wt% scaffolds with different glycerol content.

3.2 Morphology and pore structure

Fig. 2(a–e) showed the microstructure of the hybrid scaffolds with different contents of GO. All scaffolds exhibited an interconnected and continuous pore structure, but the morphology and pore diameter of the scaffolds was partly dependant on the GO content. Compared with the leaf-shape or spindle-shape structure of the single component SF scaffold, the pore structure changed more elliptical and the scaffolds exhibited an improved porous structure after GO was added. Furthermore, the average pore diameter decreased from 102 to 81 μm (Fig. 2f) and the uniformity of the pore size was enhanced with the GO content increased from 0 to 1 wt%. Freeze-drying is commonly used as a gentle dehydration method to dry porous scaffolds by sublimating the ice particles formed during the freezing process. The size of ice particles during freezing is dependant on the growing velocity of ice crystals, growing time, growing history, etc.⁴⁰ When GO was incorporated into the SF solution, the viscosity of the blend solution was increased due to the hydrogen bonding interaction formed between SF molecule and GO nanosheets. This made the free movement of water molecule become difficult and slow, and the resistance of forming larger ice particles increased accordingly. As a result, the pore diameter was decreased with the increase of GO content. The porosity of the hybrid scaffolds was 92–94%, regardless of the variables studied (Fig. 2f). Such pore structure was adequate for the migration and proliferation of cells, as well as vascularization.

Fig. 2 SEM images of the scaffolds' cross-section: (a) SF, (b) SF/GO 0.1 wt%, (c) SF/GO 0.2 wt%, (d) SF/GO 0.5 wt%, (e) SF/GO 1.0 wt%. (f) Pore diameter and porosity of the SF/GO scaffolds.

3.3 Structure analysis and mechanical properties

The crystalline structure and molecular conformation of the SF/GO scaffolds with different GO content were determined by XRD, FT-IR and DSC.

As shown in Fig. 3A, the characteristic XRD peak of GO appeared at 2θ = 10.6°, corresponding to a layer-to-layer distance (d-spacing) of 0.83 nm.³⁷ Previous studies on SF demonstrated that the main diffraction peaks of the silk I structure are at 12.2°, 19.7°, 24.7° and 28.2°, while silk II structure at 9.1°, 18.9°, 20.7° and 24.3°.¹ Both the SF sample and the hybrid samples showed obvious peaks at 9.1°, 20.7° and 24.7° (Fig. 3A(a–e)), indicating a typical silk II structure with a small amount of silk I structure. Moreover, no obvious diffraction peak at 10.6° was found in the SF/GO scaffolds' XRD patterns (Fig. 3A(b–e)), indicating GO was uniformly dispersed in the SF matrix without aggregation.

Fig. 3 XRD, FT-IR, DSC and mechanical characterization of the SF/GO scaffolds. (A) XRD curves: (a) SF, (b) SF/GO 0.1 wt%, (c) SF/GO 0.2 wt%, (d) SF/GO 0.5 wt%, (e) SF/GO 1.0 wt%, (f) GO. (B) FTIR spectra: (a) SF, (b) SF/GO 1.0 wt% and (c) GO. (C) DSC curves: (a) SF, (b) SF/GO 0.1 wt%, (c) SF/GO 0.2 wt%, (d) SF/GO 0.5 wt%, (e) SF/GO 1.0 wt%. (D) Compressive modulus and compressive stress of the scaffolds.

Fig. 3B illustrated the FT-IR spectra of the scaffolds. In the FT-IR spectrum of GO, strong absorption bands at 3389 cm⁻¹, 1728 cm⁻¹, 1630 cm⁻¹ and 1045 cm⁻¹ were indicative of O–H stretching vibration, C=O stretching vibration of carboxylic group, C=C stretching vibration assigned to sp² network of the unoxidized graphite domains, and C–O–C stretching vibration of epoxy group.⁴¹ Based on the previous studies of SF,^1,8 strong bands at 1631 cm⁻¹, 1523 cm⁻¹ and 1236 cm⁻¹ were observed, demonstrating a typical silk II conformation. This indicated that glycerol could regulate protein conformation and such results were consistent with the XRD results.

The DSC curves of the scaffolds were exhibited in Fig. 3C. All samples showed a bound water evaporation peak at around 83 °C. For SF, a small crystallization peak appeared, implying the unstable non-crystal structure was changed to the stable crystalline structure. However, the crystallization peak disappeared in the hybrid scaffold curves, revealing the formation of silk I or silk II structure prior to DSC scanning. This investigation might demonstrate that the presence of GO in the SF matrix promoted the formation of more stable crystal structure by the intermolecular forces between GO and SF molecular chains. The only one degradation peak of the scaffolds appeared at 292 °C, confirming the stable silk II structure formed in the porous scaffolds, which was corresponding with the XRD and FT-IR results.

The mechanical properties of the scaffolds were evaluated using compression test and the results were shown in Fig. 3D. The compressive modulus of the hybrid scaffolds incorporated with 0.1 wt%, 0.2 wt%, 0.5 wt% and 1.0 wt% GO were 19.14, 19.33, 21.73 and 24.32 kPa, respectively, higher than that of the pure SF scaffold (14.02 kPa). Furthermore, the compressive strength was improved with the increase of GO loading. The enhancement of the mechanical properties was related to the uniform dispersion of GO with high elastic modulus in the SF matrix and the intermolecular forces formed between the SF and GO nanosheets. Previous studies have verified that GO-coated or GO incorporated scaffolds had the ability to induce the osteogenic differentiation of mesenchymal stem cells due to the high elastic modulus and stiff of the cell culture matrices.^30–32 Therefore, we expect the mechanically rigid scaffolds with the improved compressive modulus could stimulate and promote the growth of osteoblasts and achieve desired results in the process of bone tissue regeneration.

3.4 Biodegradation

Biodegradability and appropriate degradation rate of the scaffolds is crucial for bone tissue regeneration. The degradation behavior of the SF/GO scaffolds was explored by incubating the samples in collagenase IA. The morphological changes of the scaffolds during the degradation process were shown in Fig. 4. Before degradation, the pore was intact and the scaffolds maintained an integrated porous structure (Fig. 4A–E). When exposed in enzyme solution, the morphology of all the samples exhibited a drastic change. The 3-D scaffolds could not keep their original shape and the pore began to fragment and collapse after 14 days degradation (Fig. 4a–e). With the extension of the cultivated time, the scaffolds were further corroded and lose the shape drastically at 28 days (Fig. 4a′–e′). Although the significant degradation phenomenon appeared on all the samples, the influence of GO on the biodegradability of the scaffolds could not be neglected. With the increase of the GO content, the scaffolds kept a more integral form and the porous structure could be maintained for longer time during the degradation process. The single component SF scaffold almost presented a powder form while an indistinct porous structure was still visible on the SF/GO scaffolds. These results indicated that the incorporation of GO enhanced the ability of the scaffolds to resist the enzyme degradation.

Fig. 4 SEM images of the different scaffolds cultivated in collagenase IA solution: (A–E) before degradation; (a–e) after degradation for 14 days and (a′–e′) for 28 days.

To investigate the degradation rate, the scaffolds were carefully collected at each appointed time and weighted. As shown in Fig. 5, the mass loss of all the scaffolds increased with enzyme degradation progressing. During the 28 days of degradation, the SF scaffold rapidly degraded and the mass remain was only 10.36%. However, the SF/GO hybrid scaffolds showed a slowly degradation rate, and the residual material of the SF/GO 1.0 wt% reached to 50.64%. The degradation level was dependant on the content of GO.

Fig. 5 Quantitative changes of the SF/GO scaffolds during the enzymatic degradation.

GO was a critically important factor to determine the degree of degradation of the scaffolds. The amphipathic GO nanosheets made it easy to form a combination of hydrogen bonding or some other intermolecular forces with the silk backbones with alternating hydrophilic and hydrophobic nanoscale domains, which reduce the diffusion of the enzymatic solution into the scaffolds and consequently lead to a lower degradation rate. Thus, these SF/GO scaffolds could have corresponding degradation rates to match the slow formation of bone tissue.

3.5 In vitro release studies

Simvastatin was used as the model drug to investigate the release behavior of drug molecules from SF/GO scaffolds with different GO content. As illustrated in Fig. 6, with the GO content increasing from 0 to 1 wt%, the cumulative percentages of SIM released from various scaffolds was found to be 32.97 ± 2.23%, 26.01 ± 4.92%, 25.83 ± 6.25%, 21.48 ± 5.78% and 22.16 ± 5.80% during the first two days, respectively. The initial burst was possibly ascribed to the instantaneous release of surface bound drug molecules or some weaker binding of SIM to the scaffolds. The hybrid scaffolds displayed different release profile depending on the GO loading. Compared to the SF scaffold, SIM molecules were released at a slower rate from the GO-incorporated samples. The release rate decreased with the increase of GO content in the hybrid scaffolds. Release of drug from scaffolds is reported to be governed by several factors, including nature and molecular weight of drug, degree of crosslinking density, pore size of the matrix, solvent type, etc.⁴² After the initial burst stage, the further constant release from the scaffolds was attributed to the states of association between SF chains and drug molecules. The XRD and FT-IR results had confirmed the formation of the hydrophobic silk II structure, and there might be hydrophobic–hydrophobic interactions between SF and hydrophobic SIM molecules, which lead to an increasing drug residence time in the scaffolds. Furthermore, the oxygen-containing groups on the GO sheets could form hydrogen bonds with the polar side chain groups in the SF molecular. In this case, the GO nanosheets acted as physical crosslinking points and accordingly increase the crosslinking density degree of the scaffolds. Thus, the SF/GO hybrid scaffolds showed a retardant drug release profile and these results might be beneficial to realize a better match between drug delivery and bone tissue regeneration.

Fig. 6 The release of simvastatin in the SF/GO scaffolds at pH = 7.4.

3.6 Cell viability assay

Excellent biocompatibility and low cytotoxicity are fundamental prerequisites for any kind of biomaterials. In the current study, murine MC3T3-E1 cells were selected to assess the cell response on various scaffolds. LSCM was performed to observe the distribution and attachment within the scaffolds as opposed to the surface.

MC3T3-E1 cells dispersed uniformly and intimately adhere to the wall of the porous scaffolds at 5 days of culture (Fig. 7(A–E)). They showed spindle-like shape, fully extended on the scaffolds and closely connected between each other, indicating the favorable action of the scaffolds in cell adherence and proliferation. When the culture time was prolonged to 10 day, the cells in the scaffolds proliferated significantly (Fig. 7(a–e)), and this tendency particularly performed in the hybrid scaffolds with 0.1 wt%, 0.2 wt% and 0.5 wt% GO loading. Unfortunately, the cell proliferation in SF scaffolds was slighter compared with the above-mentioned 3 kinds samples and the SF/GO 1.0 wt% scaffolds showed a relatively poor function in cell proliferation. These results indicated that moderate content of GO in the SF matrix had synergistic effect on the proliferation of MC3T3-E1 cells, the scaffold without GO or with excess GO were unfavorable for the growth of bone cells.

Fig. 7 LSCM images of MC3T3-E1 cells cultured on the SF/GO scaffolds for 5 days (A–E) and 10 days (a–e): (A and a) SF, (B and b) SF/GO 0.1 wt%, (C and c) SF/GO 0.2 wt%, (D and d) SF/GO 0.5 wt%, (E and e) SF/GO 1.0 wt%.

Additionally, to further investigate the spatial distribution of cells inside the porous scaffolds, an animation was obtained by LSCM and the 3-D images captured when the sample rotated around the Y-axis were shown in Fig. 8. The MC3T3-E1 cells showed a uniform distribution on the surface of the scaffolds (Fig. 8(a)). When the cell-seeded scaffold was rotated, the internal cells hidden inside the porous material appeared gradually (Fig. 8(b and c)), which indicated that the scaffold was able to support the cell growth and proliferation both on its surface and interior.

Fig. 8 Spatial distribution of the MC3T3-E1 cells inside the SF/GO 0.2 wt% scaffold cultured for 10 days observed from different angles. (a) 0°, (b) 30°, (c) 85°.

The SEM images of the MC3T3-E1 cells cultured on the SF/GO 0.5 wt% scaffold at day 7 were provided in Fig. 9. The cells exhibited normal morphology and adhered to the scaffold tightly. Moreover, a large amount of extracellular matrix and obvious pseudopods were found around the cells, indicating the high viability and good growth state of the cells.

Fig. 9 Morphology (7 days) of MC3T3-E1 cells cultured on the SF/GO 0.5 wt% scaffold: scale bar for a–c = 10 μm and d–f = 1 μm.

The cellular growth behaviour in the SIM-loaded scaffolds was also observed by LSCM when the cells were cultured to day 10 (Fig. 10). A typical cell adherence and good growth state were found on different porous scaffolds: cells covered the surface of the scaffolds, attached to the pore wall and exhibited the spindle-like characteristic. Compared with the samples without SIM, the cells cultured on the drug-loaded scaffolds showed stronger red fluorescence and higher cell distribution was found on the SF/SIM and SF/GO/SIM samples (Fig. 10(b and d)), indicating that the fabricated scaffolds containing SIM were non-toxic and in favour of the attachment and proliferation of MC3T3-E1 cells.

Fig. 10 LSCM images of MC3T3-E1 cells cultured on various scaffolds for 10 days (a) SF, (b) SF/SIM, (c) SF/GO, (d) SF/GO/SIM.

MTT test was used to study the cell viability and proliferation on different scaffolds. The influences of GO content and SIM release on the cell viability were both investigated. As shown in Fig. 11A, the number of cells on the scaffolds with different GO loading increased gradually during the 10 day culture, demonstrating that the SF/GO scaffolds could support the cell growth and proliferation. However, the cell proliferation in the scaffolds with 0.1 wt%, 0.2 wt% and 0.5 wt% GO content was significantly higher than that of the SF and SF/GO 1.0 wt% at day 1 to day 10 (P < 0.05). The drug-loaded scaffolds (Fig. 11(B)) showed similarly increasing cell proliferation with the extending of incubation time. However, better biocompatibility was found on the scaffolds incorporated with SIM. The cells cultured on the drug-loaded material had higher viability and faster proliferation rate than that on the scaffolds without SIM. The sustained release of SIM from the hybrid scaffolds was beneficial to the growth and proliferation of the osteoblasts.

Fig. 11 MTT assays of the cell adhesion and viability on various scaffolds. (A) SF/GO scaffolds with different GO content. (B) SF/GO scaffolds loaded with SIM.

In brief, LSCM images and MTT results showed that the cell viability in the scaffolds with moderate amount of GO was better than that of the other two kind samples. Previous study had proved that the incorporation of GO could enhance the mechanical properties of the scaffolds and consequently activate the functions of osteoblasts cultured in the scaffolds. Thus, we speculated that the improvement of the SF/GO hybrid scaffolds' compressive modulus was a key factor to promote the growth of bone cells which prefer to anchor onto stiff matrices, and the GO content was another critical reason because the cytotoxicity of GO was concentration-dependant. With regard to the drug-loaded scaffolds, the incorporation of SIM made contributions to the better cytocompatibility according to the experimental results. Although more studies are still necessary to further explore the application of such scaffolds used in bone tissue engineering, the preliminary results have demonstrated that both SF/GO and SF/GO/SIM materials have potential to serve as scaffolding options for bone tissue regeneration.

4. Conclusions

SF/GO scaffolds were prepared by lyophilization and glycerol was firstly used as a plasticizer in the SF/GO scaffold where glycerol induced the formation of silk II structure to achieve water stability. The GO incorporation decreased the pore size, enhanced the mechanical properties and improved the resistance to the enzyme solution of the hybrid scaffolds. The SF/GO/SIM scaffolds showed sustained release during 30 days, and the release rate depended on the GO concentration in the scaffolds. Furthermore, the SF/GO scaffolds were found to be non-cytotoxic, MC3T3-E1 cells could adhere and proliferate in the scaffolds and they displayed higher viability when cultured in the hybrid scaffolds with 0.1 wt%, 0.2 wt% and 0.5 wt% GO loading. Compared with the blank scaffolds, the SF/GO/SIM samples exhibited better biocompatibility, the cells cultured on them showed faster proliferation rate. Considering the satisfactory ability to support and promote the growth of osteoblasts, the SF/GO scaffold would become a competitive candidate for bone tissue engineering.

Acknowledgements

We gratefully acknowledge the financial support from Shanxi Scholarship Council of China (No. 2012-049).

Notes and references

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