Enhancing the Gelation and Bioactivity of Injectable Silk Fibroin

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Enhancing the gelation and bioactivity of injectable silk fibroin hydrogel with Laponite nanoplatelets Dihan Su, Libo Jiang, Xin Chen, Jian Dong, and Zhengzhong Shao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00891 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 19, 2016

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ACS Applied Materials & Interfaces

Enhancing the Gelation and Bioactivity of Injectable Silk Fibroin Hydrogel with Laponite Nanoplatelets Dihan Su,‡a Libo Jiang,‡b Xin Chen,a Jian Dong,*b Zhengzhong Shao*a a

State Key Laboratory of Molecular Engineering of Polymers, Department of

Macromolecular Science and Laboratory of Advanced Materials, Fudan University, Shanghai 200433, P.R. China. b

Department of Orthopaedic Surgery, Zhongshan Hospital, State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200032, China

‡

D. H. Su and L. B. Jiang contributed equally to this work.

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ABSTRACT Regenerated silk fibroin (RSF) of Bombyx mori silk fiber is a promising natural material for bone defect repair. However, lack of specific integrin and growth factor for osteoinduction significantly hinders its application in this area. In this paper, the role of Laponite nanoplatelet (LAP), a bioactive clay which can promote osteoblast growth, in the formation of RSF hydrogel as well as the various properties of RSF/LAP hybrid hydrogel was closely investigated. The results indicate that LAP could serve as a medium to accelerate hydrophobic interaction among the RSF molecules and a disruptor to limit the growth of β-sheet domain during the gelation of RSF. Rheological measurement suggests that the RSF/LAP hydrogel is injectable as it displays thixotropy in the room temperature. Proliferation and differentiation results of the primary osteoblasts encapsulated in hydrogel show that RSF/LAP hydrogel can promote the cell proliferation and enhance the osteogenic differentiation. The transcript levels for alkaline phosphatase, osteocalcin, osteopontin, and collagen type I osteogenic markers obviously improve with RSF/LAP hydrogel compared to the controls at 14 days, especially with the higher contents of LAP. Overall, the results suggest that the RSF/LAP hydrogel have the great potential to be utilized as an injectable biomaterial for irregular bone defect repair. KEYWORDS: reconstitute silk fibroin; clay; hybrid hydrogel; thixotropy; primary osteoblasts;

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1 INTRODUCTION The prevalence of tissue injuries in clinic has motivated the tissue engineering researches, especially the bone tissue engineering, aiming at the in vitro generation of the replacement. One potential material for such an application is hydrogel,1-2 as lots of efforts have been devoted to implanting cell-encapsulated hydrogel fabricated ex vivo into a body site such as bone and cartilage defects to foster the tissue restoration.3 Regarding the materials employed, regenerated silk fibroin, a kind of natural protein harvested from the domestic Bombxy mori silkworm silk (named RSF) or wild Antheraea pernyi silkworm silk (named ARSF), has been extensively studied on its applications in biomedicine. For example, RSF has been chosen to be the bone tissue replacement

because

of

its

abundant

source,

suitable

combination

of

strength/toughness, low inflammatory reactions, adjustable biodegradability and good blood compatibility.4-5 Unlike ARSF, RSF does not have special arginineglycine-aspartic acid (RGD) integrins which can promote the cell attachment and growth.6 Therefore, the promotion of cell response to the RSF biomaterials is one of the great challenges for the tissue engineering. Importantly, current trends in bone defect repair require biomaterials that are injectable and degrade slowly with new bone formation, along with functions of promoting osteogenesis. With the generation of various bionanocomposites, the incorporation of bioactive materials into polymeric tissue engineering matrices is regarded as a promising solution.7 RSF based biomaterials such as the scaffolds of RSF/chitosan/hydroxyapatite,8 RSF/TiO2,9 RSF/ceramic,10 RSF/PVA11 and even 3



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RSF/bioactive glass film12 have been investigated on the osteoblasts, chondrocyte or hBMSCs growth for bone tissue repair. The results indicated that the incorporation of the functional additives was able to enhance the bioactivity of the RSF based material. However, the major challenges still remain to these solid materials, mainly in terms of the poor processing,7, 13 unmatched shape with the irregular bone defect site as well as the insufficient osteoinductive properties. On the other side, clays have been regarded as potential candidates in up-regulating the osteogenesis for bone repair.7, 14 Mieszawska et al. investigated the montmorillonoid (MMT)/RSF composite film for the support of hBMSCs in osteogenic culture.15 Although these RSF based films were suggested to facilitate proliferation and differentiation compared with the controls, it was still considerably difficult to apply them in bone defect repair in practice. As the counterpart of natural MMT, LAP is a synthetic smectic clay. LAP has been regarded as a potential candidate with tunable properties for bone defect repair due to its rich sources of osteoinductive silica inorganic species.7, 16 Moreover, LAP presents the ability to induce osteogenic differentiation of bone hMSCs in the absence of any osteoinductive factor,13 due to the released ions from LAP which indirectly upregulate the osteogenesis.17 For example, the released Mg2+ plays a critical role in cell adhesion and differentiation.18 Although these previous studies have demonstrated the beneficial effect of LAP on osteogenesis, LAP cannot be used alone in powder. In this regard, a promising solution lies in the incorporation of LAP nanoplatelets with RSF to prepare an 4


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injectable hydrogel. In the present study, the functional RSF hydrogel mixed with LAP nanoplatelets was investigated to offset the drawbacks of LAP and RSF. The gelation mechanism as well as the properties of RSF/LAP hydrogel were explored by various techniques, such as NMR, FTIR, Raman spectroscopy and rheological test, while the proliferation and differentiation capacity of primary osteoblasts encapsulated within the RSF/LAP hydrogel were examined by the Cell Counting Kit-8 (CCK-8) assay and Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay

2 MATERIALS AND METHODS 2.1 Materials The cocoons of Bombyx mori silkworm were bought from Shandong Province, China. Synthetic Laponite XLG, Na+0.7 [(Si8Mg5.5Li0.3)O20(OH)4]-0.7, was kindly provided by Rockwood and was used after being dried at 100 oC for 4 h. The sodium polyacrylate solution was prepared by neutralizing polyacrylic acid (Mw = 3000) aqueous solutions with NaOH. The regents related with cell culture were obtained from Gibco, USA. CCK-8 was purchased from Dojindo Laboratories, Japan. Live/Dead cell viability assays were purchased from Invitrogen, USA. The real time PCR regents were bought from Takara Bio. Co. LTD, China. The other chemicals were all purchased from Aladin and used as received. Deionized water (resistivity >18.2 MΩ cm) was obtained by Millipore purification apparatus. 2.2 Preparation of RSF, LAP and RSF/LAP hydrogel 5



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The RSF aqueous stock solutions were prepared by degumming and dissolving of the Bombyx mori silk fiber as previously described.19 Briefly, cocoons of Bombyx mori were boiled for 45 min in an aqueous solution of 0.02 M Na2CO3 and then rinsed thoroughly with pure water. After drying at 40 oC, the extracted silk fiber was dissolved in 9.3 mol/L LiBr solution, at 60 oC for 1 h. Then the solution was dialyzed against deionized water for 72 h at 25 oC with a 14,000 molecular weight cut off dialysis tube to remove the salt. The acquired the RSF solution with the concentration of 4.5% (w/w) was stored at 4 oC before used. According to the dispersal method of Laponite in aqueous solution by Wang et al.,20 the presented Laponite white powder was dispersed in water under stirring for at least 24 h, then the sodium polyacrylate was added in the solution and continued stirring for another 2 h to get the modified Laponite nanoplatelets (LAP). RSF solution and LAP solution were mixed by pipette in different ratios of 0-5 wt% (LAP/RSF) at 25 oC. In order to decrease the time of RSF/LAP hydrogel formation, the mixture in a 50mL centrifuge tube were pretreated by the sonication21 for 20 s at the 20% amplitude with a FS-1200N Sonifier (1200w) (SX-Sonic. Co., Shanghai, China), and then were incubated at 37 oC. The T0 point of gelation was set at the RSF/LAP mixed solution after being sonication. The sol-gel transition was monitored by an inverted test tube method.

2.3 Characterization of the mixtures of RSF/LAP The transmission electron microscopy (TEM) is conducted on a JEOL 2100F 6


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(Japan) microscope at 200 kV. For sample preparation, the dilute LAP or RSF/LAP solution with RSF concentration of 4×10-5mg/mL was dropped on a copper grid and dried by infrared lamp. Atomic force microscopy (AFM) observations were performed on a Dimension 3100 Nanoscope IV equipped with silicon cantilevers in the tapping mode (Bruker, USA). The dilute RSF/LAP solutions were dropped onto a fresh mica substrate and allowed to fully dry in a desiccator for 24 h at 25 oC. Field-emission scanning electron microscopy (FE-SEM) was carried out on a Hitachi S-4800 (Hitachi, Japan) and the element was analyzed by the energy dispersive spectrometer (EDS) accessory. The sample was prepared by the RSF/LAP solution with the RSF concentration of 0.4 mg/mL. The rheological experiments were performed at 37±1oC in the strain controlled mode using a Physica MCR 301 rheometer (Anton Paar GmbH, Austria) with a cone-and-plate geometry of 1o incline, 60 mm diameter. The strain value and frequency value were selected in the liner region. The dynamic frequency sweep experiments of the RSF/LAP solution and hydrogel were performed in the range of 100-0.1 rad/s at 1% strain. To study the thixotropic properties of RSF/LAP hydrogel, 3000% strain for 60 s followed by 1% strain, was run at 1 rad/s. To minimize evaporation, a solvent trap with low viscosity mineral oil was employed. The temperatures of the sample and atmosphere were controlled by a Peltier temperature control device. In order to study the gelation mechanism of the RSF/LAP hydrogel, the interactions and structure change of RSF were investigated. 7


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NMR spectra were acquired with a DMX 500 spectrometer (Bruker, Switzerland) operating at 79.54 and 100.69 MHz, respectively. The cross-polarization (CP) experiments were carried out under spun at about 10 kHz, with a delay time of 5-10 ms and a contact time of 2 and 5 ms. Dynamic structure change was performed by a Nicolet FTIR 6700 spectrometer (Thermofisher, USA) and Laser Raman spectrometer with a Renishaw inVia Reflex spectrometer coupled to a Lieca microscope (Renishaw, England). The X-ray diffraction (XRD) data were recorded on an X’pert Pro with Cu Kα radiation (Bruker, Germany).

2.4 The viability of primary osteoblasts in RSF/LAP hydrogel The primary osteoblasts (Fig.S1) were isolated from skull of the new born SD rats as described previously,22 and were cultured and expanded in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in an incubator 37 oC and 5% CO2. Before use, cells were digested with 0.25% trysin-EDTA from culture flasks and resuspended in DMEM to obtain a cell density of 5×107 cells/mL. The RSF/LAP solution was sonicated at 20% amplitude for 20 seconds, and then it was filtered with a 0.22 µm filter in the clean bench immediately. After about 30 min incubation of the solution at 37 oC, 50 µL of cell suspension was added and mixed with the solution to reach a final concentration of 2×105 cells/mL. The plates were placed into the incubator and allowed to gel for an additional 0.5-1h at which time the wells were added with media. The media were changed every 3 days. All cell cultures were 8


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performed in an incubator maintained at 37 oC and 0.5% CO2. The biocompatibility of the RSF/LAP hydrogel was evaluated by using the Cell CCK-8 assay according to the ISO standard (ISO 10993.12−2004). Primary osteoblasts were cultured on the surface of those hydrogels of RSF/LAP and RSF/Laponite (as the control). After cultured for 1, 3, 5, 7 and 14 days, the hydrogel was washed with PBS solution and incubated in the CCK-8 along with cell culture media in a ratio of 1:10. The absorbance of the solution at 450 nm was tested using an Elx 800 instrument (Biotek, U.S.A.) after incubation for 2-4 h. The viability of primary osteoblast within the RSF/LAP hydrogel was examined using a Live/Dead Viability Cytotoxicity Kit. The staining solution containing 2 mM calcein AM and 4 mM EthD-1 was added directly onto the gel and allowed to react for 45 min before observation by Laser Scanning Confocal Microscope (CLSM) (Nikon C2+, Japan). For the SEM imaging, the hydrogel were fixed in 4% glutaraldehyde at 4 oC overnight, and dried by ethanol with gradient concentrations.

2.5 ALP activity and the gene expression assay The osteogenic differentiation was assessed by measuring the alkaline phosphatase (ALP) activity of primary osteoblasts. After 1 day of culture, the medium of stimulation group was changed to osteogenic differentiation medium with osteoinductive factors, and then changed every 3 days. On day 7 or 14, the samples were digested in 1 mL of papain solution (125 µg/mL papain in 0.1 M sodium phosphate with 5 mM Na2-EDTA and 5 mM cysteine-HCl at pH 6.5) for 16 h at 60 oC, 9



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and the ALP activity was measured by the BCIP/NBT ALP Color Development Kit (Beyotime Institute of Biotechnology, Shanghai, China). For the gene expression analysis, the total RNA in the cultured hydrogel was extracted at the day of 7 or 14, and PCR amplification was performed on a Mastercycler ep realplex PCR system (Eppendorf, Germany). The detailed operation was described in the supporting information.

2.6 Statistical analysis Statistical analysis was performed using ANOVA and a two-tailed Student’s t test with p < 0.05 as the criterion for statistical significance. After performing ANOVA, the differences between two groups were explored with a Turkey test, if necessary. All tests were carried out in triplicate and the data were presented as mean ± standard deviation unless otherwise mentioned.

3 RESULTS AND DISCUSSION 3.1 The dispersion of LAP in the RSF matrix Currently, it remains a great challenge to uniformly disperse the clay nanoparticles in the polymer based hybrid nanocomposites, because both thermodynamically and kineticly, the organic polymer matrices (usually hydrophobic) restrict the dispersal of the inorganic hydrophilic clay.23 Laponite nanoplatelets, as a kind of synthetic smectic clays, tend to form unpredictable structure in water because of the electrostatic attraction between their oppositely charged faces and edges.24 10


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Interestingly, Laponite could be exfoliated and dispersed homogenously in water when mixed with sodium polyacrylate, owing to the mutual negative charge repulsion caused by a possible wrapping of their positive-charged edge parts.20 Accordingly to the TEM image shown in Fig.S2, LAP nanoplatelets are evenly dispersed in the aqueous solution with a thickness of approximately 1.5 nm and lateral dimensions of 25-60 nm, which allows LAP to be homogeneously mixed with the RSF aqueous solution. To observe the dispersal of LAP with the interaction of RSF, the microstructure of the RSF/LAP blend and RSF/LAP freeze-dried hydrogel were imaged by AFM, TEM and FESEM. The AFM image (Fig.1a) shows that the LAP nanoplatelets (pointed by the red arrows) disperse well in the RSF matrix, despite a few nanoscaled aggregations with the width of about 40 nm (pointed by the green arrow). Besides, in the RSF/LAP blend, RSF tends to form the nanofibrils (pointed by the white arrows). Consistently, the RSF nanofibrils (pointed by the white arrows) as well as their aggregates (pointed by pink arrows) in RSF/LAP can also be observed in the TEM and the FESEM images (Fig.1b and 1c). Moreover, it can be seen in Fig. 1c that the LAP nanoplatelets (pointed by the red arrows) are evenly distributed in the RSF nanofibrils, which confirms that LAP are exfoliated and disperses stably and homogenously in the aqueous solution of RSF and during the casting process of the RSF/LAP mixture. On the other hand, the dispersal of LAP in the hydrogel is investigated by FESEM and EDS. According to the FESEM image (Fig.1d), normal lamellar structure 11



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was observed in the freeze-dried RSF/5%LAP hydrogel; and LAP (pointed by the red arrows) uniformly distributes on the surface (Fig. 1e). The EDS mapping results (Fig.1f) also present that the Si and Mg element of LAP are well dispersed in the samples.

Figure 1. a) AFM height image of the dilute RSF/LAP blends. b) RSF nanofibrils in RSF/LAP blend observed by TEM. c) FESEM image of RSF/LAP blends on the glass substrate. d) FESEM image of the freeze-dried RSF/LAP hydrogel. e) The zoom in image of Fig.1d. f) EDS analysis of the N, Si and Mg on the freeze-dried RSF/LAP hydrogel. The proportion of RSF to LAP was 20:1 (w/w). Red arrows: LAP; green arrow: small aggregate of LAP; white arrows: RSF nanofibrils; pink arrows: RSF fibers from RSF nanofibrils.

3.2 The influence of LAP on the gelation of RSF It is well known that the conformation of RSF, which is dominated by highly characteristic repetitive GAGAGS peptide segment, is able to spontaneously transform from a random coil/helix in the sol to β-sheet in the gel.25 Such a conformation or sol-gel transition could be accelerated by various factors such as low pH,26 high temperature,27 sonication21, alcohol treatment28, etc. For example, the 12


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sol-gel transition of the aqueous solution of RSF requires a few weeks at 4 oC, about 10 days at 37 oC and only about 40 mins at 95 oC, respectively. In the case of ultrasonic pretreatment for a short time (e.g. 20 s), the gelation time of RSF is shortened to around 4 days at 37 oC. This is because that the movement of RSF molecular chains can be significantly promoted by the thermal effect or sonication; and the fast changes in hydrophobic hydration induce rapid physical cross-linkers via the β-sheet domain in the RSF system, resulting from the energy input to the system.21

Figure 2. a) The gelation time of RSF aqueous solution (4.5 wt%) with different contents of LAP( 0-5%, w/w). b) The gelation time of RSF/LAP mixed solution after ultrasonic pretreatment for 20 s. The sample was incubated in 37 oC for the detection.

According to the previous reports, MMT, another kind of clay, is able to efficiently promote the conformation transition of RSF from random coil to β-sheet in the suitable conditions.29 It can be found that, similarly, LAP accelerates the gelation process of RSF, as the gelation time of the RSF solution incorporated with a little amount of LAP evidently decreases (Fig.2a). Moreover, after a mild sonication pretreatment for about 20 s, the RSF/LAP mixed solution presents an even shorter gelation time, decreasing from about 120 min to about 60 min with the increase of the 13



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LAP content from 1% to 5% (Fig.2b). It should be noted that the raw Laponite can also shorten the gelation time of RSF (Fig.S3), suggesting that the modification of Laponite with sodium polyacrylate only improves the dispersion of the LAP in RSF.

3.3 The rheological properties of RSF/LAP hydrogel Fig.3a shows the storage modulus (G’) and loss modulus (G’’) of the original (i.e. without sonication) RSF solution and RSF/5%LAP mixed solution, and those of the solutions pretreated by sonication as a function of frequency. It can be seen that both the pretreated RSF solution and the RSF/5%LAP mixed solution present the gel-like behavior in a certain degree. It is well known that during sonication, the RSF solution undergoes extreme local effects, such as heating, high pressure, increased air-liquid interfaces and high strain rates,30 which could affect the process of the β-sheet formation of RSF. More interestingly, the G’ of the RSF/LAP solution pretreated by sonication is larger than the G’’ and the difference is nearly frequency independent. This indicates that the gelation happens in the RSF/LAP solution just after sonication, although it has not been confirmed by an inverted test tube method yet. Fig.3b exhibits the G’ and G’’ of various RSF/LAP hydrogels as the function of frequency. It can be seen that the G’ of the RSF/LAP hydrogel increases from about 80 kPa to 200 kPa with the increase of the LAP content from 1% to 5%; and is much larger than that of pure RSF hydrogel (30 kPa). The results suggest that the incorporation of LAP in RSF can greatly improve the mechanical properties of the hydrogel.

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Figure 3. a) Frequency sweep of RSF (4.5wt%) and RSF/5%LAP solution with 1% strain, with and without the sonication. b) Frequency sweep of various RSF/LAP hydrogels with 1% strain. c) Large-amplitude oscillatory shear with strain from 1% to 3000% and shear recovery test of RSF/5%LAP hydrogel with strain at 1%. The blue curve presents the change of strain with time. d) The test of repetitive recovery of the gel. It takes 70s to recover the gel behavior after shearing with 3000% strain for 60 s.

It has been reported that the RSF hydrogel networked by nanofibrils has remarkable thixotropy.31 Therefore, the large amplitude oscillatory shear was employed to evaluate the thixotropic property of RSF/5%LAP hydrogel which may also be constituted by nanofibrils (Fig.3c). It can be found that the cross point of G’ and G’’ of the hydrogel is at about 7% of strain, suggesting the hydrogel starts to collapse in this critical strain. With the increase of the strain to 3000%, the sample is further liquefied, as both of its G’ and G’’ dramatically decrease and the G’’ is larger

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than the G’. Nevertheless, when the shear strain is switched from 3000% to 1%, the sample immediately presents the gel behavior, as its G’ becomes significantly higher than G’’ and recovers to about 82% of the original value. Indeed, the recovery rate of a thixotropic hydrogel is a crucial parameter for the practical application. Thus, the recovery rate as well as the repeatability of turnover between gel and fluid of RSF/5%LAP is examined and the results are shown in Fig.3d. The data show that the liquefied RSF/LAP can withstand a large strain treatment for 60 s and quickly recover the gel behavior within 70 s, even after continuous repeats for 6 times. Consistently, the RSF/LAP hydrogel keeps its gel properties after injected through a syringe (Fig.S4), which is favorable for fixing the irregular defects of the bone. It can be speculated that the thixotropy of these RSF based hydrogels is corresponded to the RSF nanofibrils dominated by the β-sheet conformation. During the “liquefying” under shearing strain, the domains of RSF are still crosslinked through the β-sheet structure which can hardly be destroyed by shear force. Once shearing strain is ceased, the nanofibrils tend to aggregate and restore the network of the hydrogel.

3.4 The mechanism of LAP enhancing the gelation of RSF Generally, the intensity ratio of the doublet bands around 850 and 830 cm-1 in a Raman spectrum for proteins is related to the microenvironment of tyrosine (Tyr).32 Tyr in RSF is comparatively abundant (about 5.2 mol % of the amino acid residues)33 and locates in the GA-rich segments; and therefore, it can be taken as a sensitive probe to detect the changes of the microenvironment around the Tyr-containing

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domains resulting from the conformational changes of RSF.34 As shown in Fig. 4a, the I850/I830 ratio of the RSF/LAP mixtures decreases from 2.6 to 1.6 with the increase of incubation time, indicating the migration of Tyr from a hydrophobic environment to a hydrophilic environment34-35 upon the formation of hydrogel. It has been determined that Tyr moves from the interior of RSF to a more polar environment when the conformation of RSF changes from random coil to β-sheet.36-37 Therefore, it is indicated that the conformation of RSF is dominated by random coil in the initial RSF/LAP mixed solution and then converts to β-sheet during the incubation to form hydrogel. Moreover, the conformation changes of RSF are confirmed by the changes of β-sheet contents (Fig.4b), which can be semiquantitatively calculated by the amide III deconvolution of the FTIR spectrum (Fig.S5), based on the previous report.38 As shown in Fig.4b, at the beginning of incubation, no significant difference can be observed between the β-sheet content of RSF/5%LAP and that of pure RSF. However, the β-sheet contents of RSF/5%LAP mixture dramatically increase to 36±0.5% during the time course of 60-90 min, which is most likely to be attributed to the “nucleation dependent aggregation” of RSF.39 By comparison, the conformation of RSF remains to be dominated by random coil (low β-sheet content) in the pure RSF solution for a relatively long time and eventually transfers to β-sheet along with the hydrogel formation (about 4 day’s incubation). Additionally, the contents of β-sheet in both hydrogels are relatively similar. Thus, the results of Raman and FTIR spectroscopies indicate that a) as an inorganic component, LAP accelerates the gelation of RSF via influencing the process of the conformation transition of the protein; and b) the 17



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resulting β-sheet contents of such RSF based hydrogels are not significantly affected by the addition of a certain amount of LAP.

Figure 4. a) The variation of I850/I830 in Raman spectra of pure RSF and RSF/5%LAP with the incubation time. b) The semi-quantitative changing of β-sheet content in pure RSF and RSF/5%LAP with the incubation time. c)

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Si CP MAS

NMR of Laponite and RSF/10%Laponite. d) FTIR monitoring on Laponite and RSF/10%Laponite; e, f) The deconvolution of Ala Cβ carbon (13C CP/MAS NMR) to the freeze-dried hydrogels of pure RSF and RSF/5%LAP, and inset images presenting the transparence of the related hydrogel, respectively. Region I: β-sheet conformation; Region II: random coil conformation.

In order to further investigate the role of Laponite/LAP on the conformation changes of RSF, the interactions between Laponite/LAP and RSF were analyzed in details. Although the noticeable electrostatic interaction between RSF and Laponite is able to suppose due to the amphoteric electrolyte properties of RSF and the electronegative surface of Laponite, the other interactions such as hydrogen-bonding and hydrophobic interaction between both components are rarely to be figured out, on

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account of RSF itself possesses quite strong ones.40 To confirm the hydrogen bonding between RSF and Laponite in the hybrid, the

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Si cross-polarization/magic angle

spinning (CP/MS) NMR spectra of Laponite and RSF/10%Laponite were obtained (Fig.4c), focusing on the chemical shift around -90 ppm related to Si-OH.41 It can be seen that there are two resonances which correspond to trioxo coordinated framework silicon (Q3, around -94.1 ppm) and to isolated silanol groups at the silicate sheet edges (Q2, around -84.9 ppm), respectively.42 It has been reported that the decrease of the relative intensity of Q2 signal is related to the reaction of Si-OH on the clay sheets.41 In this regard, the lowered intensity ratio of Q2 to Q3 of RSF/10%Laponite (20.7%), compared with that of Laponite (29.8%), suggests the possible formation of the hydrogen bonds between Si-OH of Laponite and -NH2/-COOH/-OH of RSF. According to the FTIR spectra shown in Fig.4d, the band of Si-O shifts from 1010 cm-1 in Laponite to 1002 cm-1 in RSF/10%Laponite, (similar results are also obtained for LAP and RSF/5%LAP, as shown in Fig. S6), which confirms the hydrogen bonding between RSF and Laponite/LAP.43 Therefore, it is suggested that both electrostatic interactions and hydrogen bonding between Laponite/LAP (which is considered as the medium) and RSF facilitate the spatial presentation of hydrophobic segments and promote inter- and/or intra-molecular hydrophobic interactions, resulting in an accelerated gelation with an increase of β-sheet contents. It is worth noting that RSF/5%LAP hydrogel is less opaque than pure RSF hydrogel, although they have the similar β-sheet contents determined by different techniques such as 13C CP/MAS NMR44 (68 % vs. 64 %, Fig.4e and Fig.4f) and FTIR 19



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(37 % vs. 36 %, Fig. 4b), suggesting that the transparence of RSF hydrogel is likely to be associated with the size of the β-sheet domains rather than the β-sheet content. XRD patterns of both freeze-dried hydrogels (Fig. S7) provides further evidence: the pure RSF presents a sharper peak around 20.1o (the characteristic peak attributed to β-sheet) than RSF/5%LAP. According to the peak broadening analysis with classic Scherrer equation about the relationship between the half-peak width and the grain size,45 the grain size of the β-sheet domain in RSF/5%LAP is smaller than that in pure RSF. When the RSF/LAP aqueous solution is incubated at 37 oC after slight sonication (20% amplitude, 20s), the interactions including hydrogen bonding, electrostatic interaction and hydrophobic interaction between RSF and LAP may facilitate the spatial presentation of the adjacent GAGAGS segments at early stage, and then escalate the number of the β-sheet domain which plays the role of cross-linker in the hydrogel. With the formation of those multitudinous smaller β-sheet domains, the RSF/LAP hydrogel exhibits less opaque and better mechanical properties than the pure RSF hydrogel. Overall, it is suggested that LAP is most likely to serve as a medium to accelerate the hydrophobic interactions among RSF and as a disruptor to limit the growth of β-sheet domain during the gelation of RSF.

3.5 Cell proliferation on the RSF/LAP hydrogels The surface topographies on primary osteoblasts’ spreading and the morphology of various RSF/LAP hydrogels were preliminarily analyzed by microscopic 20


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observation at different times. After culturing for 1 day, the primary osteoblasts with elongated and flattened morphology are observed in all RSF/LAP hydrogels studied, standing out in the sample of RSF/5%LAP, compared with those less elongated cells on the pure RSF hydrogel (Fig.5a). After 7 days’ culturing, more cells can obviously be observed on RSF/LAP than on pure RSF hydrogel. Consistently, according to the proliferations of primary osteoblasts by day 7 and day 14 (Fig.5b), the cells on RSF/LAP hydrogels and even on RSF/Laponite hydrogels significantly outnumber those on pure RSF. These results indicate that LAP/Laponite facilitates the primary osteoblasts proliferation on the surface of the RSF based hydrogels. In order to further examine the cell viability in the hydrogel, primary osteoblasts encapsulated in the RSF/1%LAP hydrogels were monitored by Live/Dead assay within 2 weeks. As shown in Fig.6a, the live primary osteoblasts showing green fluorescence are always the predominant population which is positively correlated to the culturing time. This indicates that RSF/1%LAP hydrogel provides proper microenvironment for cell growth. The viability of the primary osteoblasts in RSF/LAP hydrogel with different contents of LAP were also investigated by the three dimensional reconstruction of CLSM. After culturing for 7 days, primary osteoblasts grow throughout the hydrogels; and much more live primary osteoblasts are observed in RSF/1%LAP and in RSF/5%LAP hydrogel than in pure RSF hydrogel (Fig.6b). This result is in agreement with the previous report that LAP plays an important role in facilitating the adhesion and the proliferation of cells.46 Consistently, the FESEM images show the similar results: much more dehydrated primary osteoblasts with 21



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spherical morphology are observed in RSF/LAP hydrogels than in pure RSF hydrogel (Fig.6c).

Figure 5. a) The primary osteoblasts spreading situation on the surface of RSF/LAP hydrogels. b) The proliferation of primary osteoblasts in various hydrogels for indicated days (detecting with CCK-8 analysis). Value was presented with mean ± SD, *P

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