Functionalization of Silk Fibroin Electrospun Scaffolds via BMSC

Feb 19, 2019 - The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 , People's Republi...
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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Functionalization of Silk Fibroin Electrospun Scaffolds via BMSC Affinity Peptide Grafting through Oxidative Self-Polymerization of Dopamine for Bone Regeneration Jiannan Wu,†,‡,⊥ Lingyan Cao,†,‡,⊥ Yang Liu,§ Ao Zheng,†,‡ Delong Jiao,†,‡ Deliang Zeng,†,‡ Xiao Wang,†,‡ David L. Kaplan,∥ and Xinquan Jiang*,†,‡

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Department of Prosthodontics and ‡Shanghai Engineering Research Center of Advanced Dental Technology and Materials; Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology; National Clinical Research Center for Oral Diseases, Shanghai Ninth People's Hospital, College of Stomotology, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China § The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China ∥ Department of Biomedical Engineering, 4 Colby Street, Tufts University, Medford, Massachusetts 02155, United States ABSTRACT: Electrospun scaffolds have been broadly studied to enhance bone regeneration because of the ability to simulate the structure and biological functions of the extracellular matrix. Polydopamine (PDA) is used to coat various surfaces at a slightly basic pH (8−8.5) and spontaneously reacts with nucleophilic functional groups. It is suitable for surface modifications of scaffolds correlated with bone formation. E7 is a newly discovered peptide with specific affinity for bone marrow mesenchymal stem cells (BMSCs). It can be useful for recruiting stem cells. Here, electrospun silk fibroin (SF) scaffolds were fabricated, and PDA was used for surface modification followed by grafting E7 (SF−PDA−E7). These composite SF−PDA−E7 electrospun scaffolds improved hydrophilicity, facilitated cell proliferation and adhesion, and boosted the osteogenic differentiation of BMSCs by creating osteoinduction conditions under the synergistic effects of PDA and E7. Moreover, the scaffolds showed high efficiency for recruiting BMSCs induced by E7 both in vitro and in vivo, which was associated with the SDF-1α/CXCR4 axis and the p38, extracellular signal-related kinase, and Akt signal transduction pathways. These functionalized electrospun scaffolds promoted regeneration of bone in the rat calvarial bone defect model. In general, this study verified that PDA could be a simple and efficient method for surface modification, and E7grafted PDA-modified SF electrospun scaffolds were suitable for bone tissue engineering. KEYWORDS: electrospun scaffold, dopamine, E7 peptide, silk fibroin, bone regeneration, cell recruitment, signal pathways cellular recognition sites for bone formation.9 Furthermore, the structure of these scaffolds could be designed to resemble natural mineralized bone tissues using different biomaterials collectively, for example, polymeric components and inorganic fillers.10 Existing electrospun scaffolds are primarily composed of synthetic polymer materials, and these synthetic polymers are easy to process with high degradability;7,8 however, the surface of these polymers is largely hydrophobic and lacks cell recognition sites.11 In contrast, scaffolds prepared from natural biological polymers contain active groups, which often boost cell growth and provide scaffolds with good cytocompatibility and biological activity.12,13 Accordingly, electrospun scaffolds

1. INTRODUCTION Scaffolds applied in tissue engineering have significant effects on tissue regeneration, creating a template for tissue regeneration and regulating cells’ behavior.1,2 Among different types of scaffolds generated using various processing techniques, nanofibrous scaffolds generated by electrospinning are capable of emulating the native structure and biological functions of the extracellular matrix. These systems are widely used in tissue engineering because they exhibit high surface area and porosity and can provide options for surface functionalization.3−5 A growing number of studies showed that electrospun scaffolds exhibited potential applications in bone tissue repair.6 Existing studies focused on electrospun scaffolds prepared from poly(lactic-co-glycolic acid) or polylactic acid.7,8 To improve their utility in tissue regeneration, bioactive molecules were often introduced to the electrospun scaffolds to enhance biocompatibility and create © XXXX American Chemical Society

Received: December 19, 2018 Accepted: February 5, 2019

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DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

The recruitment of stem cells to tissue defect sites is associated with various cytokines and receptors in the body.38,39 Stromal cell-derived factor-1 (SDF-1α) is a small molecular weight cytokine produced by MSCs and belongs to the CXC chemokine family. CXCR4 is the only transmembrane receptor that mediates the function of SDF-1α, and it is expressed on the surface of stem cells presented in peripheral blood and bone marrow.40,41 According to recent studies, the interaction of the SDF-1α/CXCR4 not only mobilized and recruited BMSCs to target damaged tissues, but also improved paracrine function and promoted adhesion and tissue repair.42,43 It is generally known that p38, extracellular signal-related kinase (ERK), and Akt signal transduction pathways have major roles in regulating cell migration and differentiation.44−46 Accordingly, it was speculated that the vital role of E7 peptide in regulating the recruitment of BMSCs was to primarily activate the SDF-1α/CXCR4 axis and then to regulate cell biological function through p38, ERK, and Akt signal transduction pathways. In the present study, SF scaffolds were coated with PDA to use these active functional groups to graft the short E7 peptide onto the electrospun scaffolds. The process was simple and required no addition of other chemical grafting reagents. Due to the presence of E7, the composite electrospun scaffolds efficiently recruited BMSCs in situ, which effectively promotes tissue regeneration. The modified SF scaffolds with E7 immobilization via PDA linking (SF−PDA−E7) showed excellent biocompatibility, and they could promote cell adhesion and proliferation while maintaining the physical and chemical properties of the SF scaffolds. Besides, the fact that the SDF-1a/CXCR4 axis contributed to the recruitment of BMSCs induced by E7 peptide via the p38, ERK, as well as Akt signal transduction pathways was revealed in our research. According to the results, these modified SF scaffolds could effectively promote tissue repair and regeneration.

prepared from natural polymers have broad applicability and continue to arouse attention from researchers regarding bone regeneration. Silk fibroin (SF) is a natural macromolecule that is nontoxic and nonstimulatory, exhibiting good biodegradability and biocompatibility.14 SF has been widely used in tissue engineering and regeneration because of its excellent physiochemical properties. Several studies showed that SF scaffolds could serve as an artificial bone substitute with high mechanical stability and durability.15,16 Though the advantages of SF are generally known, it has several limitations on its usefulness in such applications, for example, low rates of osteoinduction. Most importantly, SF modification has primarily focused on chemical reactions to the amino acids in the silk proteins,17 and usually it involves direct mixing with other carriers for the synthesis of composite materials that promote bone restoration.18−20 To broaden the applications of SF electrospun scaffolds in tissue repair, an oxidative self-polymerization of dopamine (DA)-based surface modifying approach was used here other than other reported methods. DA is a molecule produced by marine mussels capable of aggregating into polydopamine (PDA) coatings on surfaces at a slightly basic pH (8−8.5) through oxidative self-polymerization.21,22 As PDA is adherent to the surface of a material, options are provided to regulate interactions between materials and cells on various surfaces.23,24 Since PDA has functional groups on its surface, for example, catechols and amino groups, it offers useful surface activity to nucleophilic functional groups.25 In several studies, PDA served as a carrier for the sustained release of drugs.26,27 For instance, Ma et al. fabricated dual-responsive capsules by modifying the surface of polydopamine-coated capsules, which exhibited excellent modifiability and high drug-loading potential of PDA. Moreover, PDA is able to convert nearinfrared light energy into heat energy and selectively kill cancer cells. Thus, PDA nanoparticles can act as very attractive photothermal therapeutic agents, and this particle has been extensively studied in the synergistic treatment of chemotherapy and photothermal therapy. Furthermore, PDA effectively promotes the adhesion and spread of cells, and there is also evidence that the DA receptor and DA signaling pathway can affect the osteogenic activity of cells.28−30 According to these characteristics, the transplantation of certain bioactive compounds or proteins onto SF electrospun scaffolds using PDA without the need for other intermediates would be useful. This strategy can be used to exploit the bioactivity of PDA to bring specific biological functions to scaffolds and help avoid defects caused by more complicated or less facile modifications. The application of the bone marrow mesenchymal stem cells (BMSCs) and the recruitment of endogenous stem cells to damaged sites have always been a research focus.31,32 In the presence of bone damage, BMSCs in the damaged tissue and circulation will migrate to the damaged site and disperse into specific tissues.33 The use of chemokines in situ helps recruit more stem cells to the damaged area and promotes the proliferation of cells that help repair tissue defects.34 A peptide sequence (E7) containing seven amino acids was identified using phage display technology, allowing for the recruitment of BMSCs both in vitro and in vivo. It also promotes adhesion and proliferation in the recruited stem cells.35−37 This response suggests that it is likely to combine a scaffold with this peptide to create a composite that helps promote cell recruitment and tissue repair.

2. MATERIALS AND METHODS 2.1. Electrospinning. The electrospinning process was developed as previously reported.47 In brief, the electrospinning device was outfitted with high-voltage equipment and coupled with a sophisticated injection pump; the collector was a tin-foil plate. The electrospinning solutions were loaded into a 2 mL syringe installed on a circular metal needle. The voltage between the needle and the ground was 20 kV. The electrospinning solutions were fed at a speed of 0.02 mL/min. Before application, all the scaffolds were dried for 24 h in a vacuum at 37 °C. 2.2. Synthesis of the Electrospun Scaffolds. All solutions were filter sterilized, and the SF scaffolds were soaked in 75% alcohol overnight. The SF scaffolds were covered with PDA by being incubated with 2 mg/mL DA in Tris-buffer solution (pH 8.5) in a general laboratory circumstance for 12 h. Subsequently, the PDAcoated scaffolds (SF−PDA) were washed with sterile water to remove unbound residues. The BMSC affinity peptide E7 (EPLQLKM; the purity was 95.64%; Scilight-Peptide Beijing, China) was conjugated on the surface of the SF−PDA scaffolds. First, the PBS containing 0.15 M NaCl was used to activate the SF−PDA scaffolds. Then, they underwent incubation with 0.1 mM E7 dissolved in Tris-buffer solution. The appropriate concentration of E7 was ascertained based on previous studies.48 The modified scaffolds (SF−PDA−E7) were washed with sterile water five times, followed by drying in a vacuum for 5 h. The SF scaffolds, SF−PDA scaffolds, and SF−PDA−E7 scaffolds were applied to subsequent experiments. 2.3. Characterization of the Electrospun Scaffolds. Under a scanning electron microscopy (SEM, Tokyo, Japan), electrospun scaffolds’ morphology was observed. The scaffolds (5 × 5 mm2) were added directly to the conductive tapes on the sample stub of the SEM B

DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

lium bromide (MTT) assay. In brief, nearly 4 × 103 BMSCs were grown on scaffolds in the 96-well culture plate for 6 and 12 h. Next, 20 μL of MTT was introduced to each well and underwent 4 h incubation after discarding the medium, and then the solution was carefully absorbed. Afterward, dimethyl sulfoxide was introduced, and the solution underwent incubation for 10 min with shaking to fully dissolve the resultant formazan. The absorbance at the wavelength of 490 nm (OD490) was measured with a universal microplate spectrophotometer (Thermo LabSystems, Beverly, MA). 2.9. Cell Viability. A live/dead assay system (Sigma-Aldrich) was used to assess the cellular compatibility of the three scaffolds. The cytoplasm of viable cells was stained in green by calceinacetoxymethyl (AM), while propidium iodide (PI) was employed for staining the nuclei of inactive cells in red. The staining solution was made proportionate by mixing calcein-AM and PI according to instructions. When the BMSCs were successfully cultured for 24 h with scaffolds, we removed the medium and washed out scaffolds with assay buffer. Subsequently, 100 μL of staining solution was introduced to each scaffold and underwent incubation for 20 min at 37 °C. After being stained, the scaffolds were observed under laser scanning confocal microscopy. The proliferation of cells was studied using an MTT experiment as described above. The cells were cultured on scaffolds with 96-well culture plates for 1, 3, 5, and 7 days. 2.10. In Vitro Cell Recruitment. Total rat bone marrow was collected and cultured with scaffolds. After 3 day culture, the scaffolds were rinsed to purge any unattached cells. Then, FITC-phalloidin staining for the cell skeleton was performed to identify the cells. Furthermore, following the instructions of the manufacturer, CD44 immunofluorescence staining was carried out. The cells cultured on scaffolds were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Subsequently, the samples were blocked by 2% bovine serum albumin (Sigma-Aldrich), and they underwent incubation with rabbit polyclonal anti-CD44 antibody (ab157107, Abcam, Cambridge, MA) at 4 °C overnight. Afterward, the samples were treated at ambient temperature for 1 h with Alexa Fluor 594conjugated secondary antibodies. Due to the strong adsorption of 4′,6-diamidino-2-phenylindole (DAPI) by the scaffolds, the cell nuclei were not seen. The scaffolds were examined by laser scanning confocal microscopy, and the density of the recruited cells on the scaffolds was calculated based on the obtained images. Each group was investigated in triplicate, and three random views were captured for each sample. Then, the recruitment of BMSCs was measured by a transwell-migration assay. The three groups of scaffolds were introduced to a transwell insert’s lower chamber, and nearly 5 × 103 BMSCs per well were cultured on the transwell insert’s upper chamber in 0.5 mL of DMEM for 12 h. Next, the transwell chamber was fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. The BMSCs not penetrating the filters were swabbed with cotton swabs, and the cells migrating to the lower surface of the filters were examined by using a microscope. The cell number was calculated from the photographed images. Each group was investigated in triplicate, and three random views were captured for each sample. 2.11. In Vitro Osteogenic Differentiation. BMSCs were seeded on the different scaffolds (diameter: 15 mm) at a density of 4.0 × 104 cells/mL and cultured in osteogenic medium containing H-DMEM supplemented with 0.1 μM dexamethasone, 50 μM ascorbic acid, 10 mM β-glycerol phosphate, and 10% FBS. BMSC-seeded scaffolds underwent culture in H-DMEM containing 10% FBS as a control. All samples were replaced with a new culture medium every 3 days. 2.12. Alkaline Phosphatase (ALP) Activity Assay. After 7 and 14 days, the ALP activity was measured with the p-nitrophenylphosphate (pNPP) liquid substrate system. In brief, the samples were treated with lysis buffer, and supernatants were collected. Subsequently, 20 μL of each lysed sample was put into a 96-well culture plate, and 100 μL of pNPP reaction liquid was poured and allowed to stand for 30 min at ambient temperature under the protection from light condition. Next, 80 μL of 1 mM NaOH was introduced to terminate the reaction, and the absorbance at the wavelength of 405 nm (OD405) was measured. Afterward, 10 μL of lysis solution was used to measure the protein concentrations by a

and coated with gold within 60 s using a gold sputtering device (SC7620, Quorum Technologies, U.K.). Chemical elements on the surface of the scaffolds were identified under X-ray photoelectron spectroscopy (XPS; Thermo, Waltham, MA). The survey spectra ranged from 1300 to 0 eV, and the pass energy of 50.0 eV was selected as the parameter. All spectra were calibrated based on the peak value of hydrocarbon C 1s (284.6 eV). The surface hydrophilicity of the scaffolds was assessed by the measurement of the water contact angle (JGA-360A, Chengde Chenghui testing co., China). The droplets in each sample showed four different points, and a charge-coupled device camera (KGV-5000, Japan) was used to capture the images. Using the device’s software, this study analyzed the results. 2.4. Molecular Docking Analysis. The molecular docking between SF to PDA and PDA to E7 peptide was further assessed using Schrodinger software, The SF crystal structure (PDB ID: 3UA0) was downloaded from the RCSB Protein Data Bank. The PDA trimer followed the possible structure of PDA reported previously.49 The three-dimensional structure of the peptide was designed using Schrodinger software. The results were assessed in line with the docking energy scores and interaction force with each other. 2.5. Quartz Crystal Microbalance with Dissipation (QCM-D) Experiments. The relative quantification between PDA to SF and PDA to E7 peptide was monitored by the QCM-D experiments (QSense, AB, Göteborg, E4, Sweden). After the stable baseline of Trisbuffer solution was achieved, DA solution was poured into the measurement chamber, and DA was oxidized and self-polymerized on the gold- and silica-coated crystals to form PDA. Subsequently, the unstable PDA was washed out with Tris-buffer solution. Next, the SF solution or the E7 peptide solution flowed into the chamber to fully react with PDA. After the reaction became stable, the incompletely linked molecules were washed out with the Tris-buffer solution readded, and the subsequent results were observed. The frequency shift (Δf) was associated with the adsorbed mass (Δm) based on the Sauerbrey equation. Δm = Δf × C/n. (C = 17.7 ng/cm2 H Z−1 at f n = 5 MHz; n = 1, 3, 5,...). 2.6. Thermogravimetric Analysis (TGA) Experiment. The characteristics of pure SF, pure PDA, and SF−PDA scaffolds were detected with TGA instrument (Q50 V20.10 Build 36). The experiment was performed in a nitrogen environment in a dynamic mode with a heating rate of 10 °C every minute; the temperature range was 30−600 °C. 2.7. Isolation and Culture of Cells. Rats (3 week-old, 100−120 g) were anesthetized with 2% pentobarbital sodium, and under sterile conditions, their tibias and femurs were removed. Under 10% fetal bovine serum (FBS; Gibco) conditions, researchers cultured BMSCs into a 25 cm2 cell culture dish with high glucose Dulbecco’s modified Eagle’s medium (H-DMEM). Then, they underwent incubation in 5% CO2 at 37 °C. To remove any unattached cells, the medium was replaced after the first 2 days. Subsequently, this replacement was performed every 3 days until 70−80% confluence was achieved. Cells between the 4th and 6th passages were applied for subsequent experiments. The procedures regarding animals here were overall approved by the Committee of Experimental Animal Administration of Shanghai Ninth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, following international ethics guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. 2.8. Cell Adhesion. After cells were seeded onto the three groups of scaffolds for 12 h, all the samples were taken out and fixed with 4% glutaraldehyde at low temperature for 4 h. The researchers dehydrated scaffolds with several gradient ethanol solutions and airdried them at ambient temperature. They coated the dried samples with gold and observed them with SEM. In addition, cell morphology after fluorescein isothiocyanate (FITC)-phalloidin staining was observed under laser scanning confocal microscopy (Leica, Wetzlar, Germany). After 12 h cell culture, the scaffolds were immersed into 4% paraformaldehyde and incubated with FITC-phalloidin for 1 h at 37 °C to observe the cytoskeleton. Evaluation of early cell adhesion was studied by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoC

DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces bicinchoninic acid (BCA) protein assay (Thermo Scientific, MA). The relative ALP activity was denoted as the ratio of OD405 to the total protein content. Each sample was analyzed in triplicate. 2.13. Mineralization with Cetylpyridiniumchloride (CPC) Assay. After 14 and 21 days, the samples were fixed with 75% ethanol, and 40 mM Alizarin Red was added at room temperature for 30 min. Subsequently, the samples were washed with deionized water five times, and 10% (v/w) cetylpyridiniumchloride (CPC; SigmaAldrich) in sodium phosphate (pH 7.0) was added and allowed to dissolve for 15 min. Next, all samples’ absorbances were measured at 570 nm. The analysis of every sample was made three times. 2.14. Immunofluorescent Staining. The bone-specific proteins of osteocalcin (OCN) underwent immunofluorescent staining, and the method was the same as previously described.50 The BMSCs were cultured on scaffolds under osteoinductive condition for 14 days and then fixed in 4% paraformaldehyde solution. Besides, the mouse monoclonal anti-OCN primary antibody (sc-390877, Santa Cruz Biotechnology, Dallas, TX) and Alexa Fluor 594-conjugated secondary antibody were employed. The samples were observed under a fluorescence microscope (Olympus IX 71, Olympus, Tokyo, Japan). 2.15. Real-Time Polymerase Chain Reaction (RT-PCR). The BMSCs were cultured on the scaffolds with osteoinductive medium for 7, 14, and 21 days. And then, the gene expression was analyzed by RT-PCR. Briefly, total RNA of the BMSCs grown on the scaffolds was harvested by Trizol (Sigma-Aldrich). The cDNA was produced by the extract RNA with the synthesis kit (Takara Bio, Shiga, Japan), and the cDNA sequences were employed for RT-PCR to assess the mRNA levels of Col-I, OCN, Runx2, ALP, and β-actin. With the 2−ΔΔCT method described, the researchers studied gene expression data.51,52 All gene expression data were normalized to that of β-actin and denoted as the fold ratio compared with that of the SF scaffolds. The following primers were applied: ALP, forward 5′-GGGGTCAAAGCCAACTACAA-3′, reverse 5′-CTTCCCTGCTTTCTTTGCAC-3′; Runx2, forward 5′-GCCGGGAATGATGAGAACTA-3′, reverse 5′GGACCGTCCACTGTCACTTT-3′; Col-I, forward 5′AATGGTGCTCCTGGTATTGC-3′, reverse 5′-GGTTCACCACTGTTGCCTTT-3′; OCN, forward 5′-GAGGGCAGTAAGGTGGTGAA-3′, reverse 5′-GTCCGCTAGCTCGTCACAAT-3′; β-actin, forward 5′-CTAAGGCCAACCGTGAAAAG-3′, reverse 5′-TACATGGCTGGGGTGTTGA-3′. 2.16. Molecular Mechanism Detection in Vitro. The molecular docking processes between E7 peptide to SDF-1α and E7 peptide to CXCR4 were also assessed using Schrodinger software, and the SDF-1α crystal structure and CXCR4 crystal structure (PDB ID: 2K04) were downloaded from the RCSB Protein Data Bank. In addition, seeding about 4.0 × 104 BMSCs was carried out in a 24-well plate until they fully adhered to the well plate. Subsequently, the three groups of the sterile scaffolds were introduced and cocultured with the cells for 3 and 7 days. The RT-PCR was used to detect the activation and the gene expression of SDF-1α/CXCR4. The experimental method has been described above. The CXCR4 and SDF-1α primers were used: CXCR4 forward 5′-AGTGACCCTCTGAGGCGTTTG3′, reverse 5′-GAAGCAGGGTTCCTTGTTGGAGT-3′; SDF-1α forward 5′-GAGCCAACGTCAAACATCTGAA-3′, reverse 5′TCCAGGTACTCTTGGATCCACTTTA-3′. Then, protein expression of the CXCR4 was evaluated by Western Blot. Total proteins from cells were extracted using RIPA buffer (BEOOTIME biotechnology, Shanghai, China). The BCA Protein Assay Kit purchased from Thermo Scientific (MA) was used in quantitative analysis of the protein concentration of cell extracts according to the kit’s instructions. About 20 μg of protein underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting on poly(vinylidene difluoride) membranes. The membranes were incubated using 5% skimmed milk under ambient temperature for 2 h; the primary antibodies of mouse monoclonal anti-CXCR4 (sc53534, Santa Cruz Biotechnology, Dallas, TX) and β-actin were incubated for 12 h at 4 °C. The membrane was rinsed with PBS three times; the second antibody labeled with horseradish peroxidase was incubated in ambient temperature lasting 1 h. Chemiluminescence

was used to visualize the bands using the excellent chemiluminescent kit (Thermo Scientific, MA). The secretion of SDF-1α was ascertained using the enzyme-linked immunosorbent assay (ELISA) kit (Cloud-clone Corp.) at the same time points. The protein content of SDF-1α was calculated in line with the absorbance value of the standard sample. The expressions of CXCR4 and SDF-1α were also tested by immunofluorescence staining. The staining methods of cytoskeleton, SDF-1α, and CXCR4 were the same as before. We used mouse monoclonal anti-CXCR4 (sc-53534, Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-SDF-1α (sc-74271, Santa Cruz Biotechnology, Dallas, TX) as the primary antibody, while Alexa Fluor 594-conjugated goat antimouse and Alexa Fluor 647-conjugated goat antirabbit were used as the secondary antibody. The nucleus and cytoskeleton were indicated by DAPI and FITC-phalloidin, respectively. In addition, a transwell-migration assay was used to verify the effect of the SDF-1α/CXCR4 axis on the E7 recruiting cells in vitro. The operation steps were as described above. Three different groups of culture media were added into the lower chambers: (A) normal medium, (B) normal medium with SF−PDA−E7 scaffolds, and (C) normal medium with SF−PDA−E7 scaffolds containing 20 nm/L AMD3100, a specific peptide antagonist of CXCR4. Besides, we assessed the effects of the SDF-1α/CXCR4 axis on p38, ERK, and Akt signal transduction pathways. The BMSCs were seeded on the well plate and cocultured with the scaffolds. The experiment was divided into three groups similar to the transwell-migration assay. Western Blot was used to ascertain protein expression following the same procedure as before. The primary antibodies against, p38, ERK, Akt, p-p38, p-ERK, p-Akt, and β-actin were incubated overnight at 4 °C. The p38 (#9212), ERK (#4695), Akt (#4691), p-p38 (#4511), pERK (#4370), and p-Akt (#4051) were purchased from Cell Signaling Technology (Cambridge, MA). 2.17. In Vivo Scaffold Transplantation. Twelve Sprague− Dawley rats (male, 6 week-old, 200 g average body weight) were randomly stratified into three groups (four animals per group) for in vivo experiments. After being anesthetized with 3% pentobarbital, the bilateral cranial bone was fully exposed to develop a round fullthickness defect with a diameter of 5 mm on either side of the midridge. Scaffolds from one of the three groups, SF, SF−PDA, or SF− PDA−E7, were implanted into the defects. Two animals underwent the same operation without scaffold implantation as the control group (blank). 2.18. In Vivo Cell Recruitment. On day 7 postimplantation, four scaffolds in each group except the control group were collected, washed with PBS, and frozen in optimal cutting temperature compound. The immobilized scaffolds were sliced into 5 μm sections and fixed with 4% paraformaldehyde. FITC-phalloidin and CD44 immunofluorescence staining processes were performed according to the description earlier, and sections were observed under laser scanning confocal microscopy. 2.19. Microcomputed Tomography (Micro-CT). During 8 weeks postimplantation, the remaining rats (two animals per group) were sacrificed, and skull samples were collected and fixed in 4% paraformaldehyde. Micro-CT (SCANCO Medical AG, Brüttisellen, Switzerland) was applied to scan skulls. The three-dimensional graphics of samples were reconstructed through image layers using VGS Studio Max software. Cylinders nearly 1 mm in height and 5 mm in diameter around the damaged areas were used as the target area. To analyze bone formation within the target area quantitatively, the researchers calculated the ratio of the regenerated bone (rb) volume to the total volume (BV/TV). The area of bone regeneration in the defect was quantified with ImageJ software. 2.20. Histological Analysis. For histological analysis, samples collected during 8 weeks postimplantation were fixed and soaked in 15% ethylenediaminetetraacetic acid. The decalcification liquid was replaced daily until the sample could be embedded in paraffin. The center of the repair site was sliced into 5 μm sections followed by hematoxylin and eosin (H&E) staining and Masson trichrome staining, as previously described.53 Finally, under a microscope, the stained sections were observed. D

DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Characteristics of the electrospun scaffolds. (A) Representative SEM images (scale bars: 2 μm); blue dashed line indicates the magnified view of the fiber surface (scale bars: 500 nm); (B) XPS spectra analysis and the corresponding (C) quantification of atomic chemical composition on the surface of the different scaffolds; (D) contact angle assay and (E) quantitative analysis of the scaffold surface (n = 3, *p < 0.05 compared to the SF group). 2.21. Statistics. Data were overall denoted as the mean ± standard deviation; p < 0.05 was deemed to be of significance. Comparisons between different groups were drawn by a paired t-test.

biomineralization.56 Besides, it has been reported that vascular scaffolds were prepared by using SF as the matrix material, and the tubular scaffolds prepared using the electrospun method exhibit very high porosity, close connection between fibers, strong explosion resistance, and similar structure to human blood vessels, which facilitated vascular construction.57 Moreover, some studies have been conducted on skin repair by modifying SF scaffolds.58 All these suggested the promising application of SF. However, SF is primarily composed of nonreactive amino acids, and chemical modifications of SF can limit some applications.17 Given this, PDA-conjugated SF scaffolds were adopted for indirect modifications. PDA has active functional groups that can interact with bioactive molecules, thereby serving as a loading system or slow-release matrix.25,26 Functional molecules (e.g., drugs or growth factors, silver nanoparticles, and proteins) can be firmly bound on the

3. RESULTS AND DISCUSSION The biomaterials applied for tissue regeneration should accelerate tissue repair and promote cell growth and functional differentiation.13,54 Study on biodegradable electrospun scaffolds and their applications has aroused increasing attention because of their excellent permeability and conducive features for cell adhesion, proliferation, and differentiation, which create a suitable microenvironment for tissue repair.3,55 SF is an excellent biomaterial applied in tissue engineering. This protein has several advantages, including plentiful supplies and biodegradability, while containing sufficient amino acids containing carboxyl groups, which are vital for the regulation of E

DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. Quantitative determination. (A) Molecular docking simulation of the PDA with silk fibroin and E7 peptide; (B) buildup of a PDA coating film in the QCM-D and the real-time frequency shift (Δf) of the QCM-D as a function of time in the presence of silk fibroin and E7 peptide; (C) thermogravimetric analysis results of pure SF, pure PDA, and SF−PDA.

the diameter of the fibers in the three groups of scaffolds did not vary significantly during the modification, suggesting the conditions were suitable with this substrate. Besides, PDA aggregates were demonstrated to be located on the surface of the scaffolds. To confirm the successful biofunctionalization of the SF scaffolds, the surface elemental composition of the three groups was determined. Due to the presence of amine bonds and amide groups between SF and PDA fibers, the nitrogen peak (N 1s, 399.8 eV) of SF−PDA scaffolds was significantly increased according to the XPS spectra (Figure 1B,C). According to the morphology of scaffolds based on SEM and the surface elemental composition of scaffolds based on XPS, PDA exhibited sufficient oxidative self-polymerization capabilities and could be combined with the SF fibers. A further elevation of the nitrogen peak in the SF−PDA−E7 scaffolds demonstrated the effective chemical bonding between the peptide and PDA (Figure 1B,C). This result revealed that the PDA and E7 peptide were successfully fixed on the SF scaffold, so the coating of PDA was proven as an effective and a relatively simple modification for transplanting the bioactive

surface of materials coated by PDA. PDA is characterized by stronger binding force, more uniform structure, and higher stability compared with chemical methods.59 Also, the surface modified by PDA shows good compatibility with chondrocytes, endothelial cells, osteoblasts, and stem cells, which facilitates cell differentiation and supports the application of materials in tissue engineering, for example, cartilage repair, vascular repair, and bone repair.60 E7 is a newly discovered peptide with a specific affinity for BMSCs, which can be used for the recruitment of BMSCs.35,36 The treatment of tissue defects by stem cell recruitment can shorten the repair cycle.61 In our study, the exterior surface of SF scaffolds was functionalized with PDA, and E7 was then grafted onto the scaffolds through the PDA, generating a new type of biological material that could recruit autologous stem cells and promote bone defect repair. 3.1. Characterization. The SF scaffolds, SF−PDA scaffolds, and SF−PDA−E7 scaffolds retained their original structures and exhibited stable nanofibrous and porous surface morphologies and structures (Figure 1A). Most importantly, F

DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

of SF−PDA scaffolds was observed with the rise of temperature, and the residual weight was about 46.4% at 600 °C. While the PDA’s residual weight was about 41.8%, and that is because of the decomposition of catechol and amino groups. The results showed that the content of PDA in SF−PDA scaffolds was about 47.1%. According to the above research findings, the quantitative relationship among SF, PDA, and E7 was preliminarily understood, and the deposition of dopamine could be accurately controlled by changing the coating time under appropriate conditions. 3.3. Cell Morphology Observation. Biological materials qualified to be used as scaffolds must exhibit good cytocompatibility.62 Thus, the adhesion of cells on the scaffolds was observed. After being cultured for 12 h, the cells were attached to the three groups of scaffolds, as determined by SEM (Figure 3A). The cells attached to the SF scaffolds did not fully spread and exhibited a triangular or long fusiform shape. However, the cells showed better spreading on

factor onto the scaffolds through their amino groups. The surface hydrophilicity of the electrospun scaffolds was also assessed (Figure 1D). The water contact angles were 85.3 ± 3.9° for the SF scaffolds, 41.6 ± 2.7° for the SF−PDA scaffolds, and 31.1 ± 1.8° for the SF−PDA−E7 scaffolds (Figure 1E). Through the addition of PDA and E7 to the SF scaffolds, the water contact angle decreased, suggesting that PDA and E7 improved the hydrophilicity. This is also in line with expectations because PDA and E7 contain many hydrophilic functional groups, for example, amino and hydroxyl groups. 3.2. Quantitative Determination. DA aggregated into PDA at a slightly basic pH (8−8.5) and was coated on the surface of SF scaffolds, and PDA contained many active functional groups, for example, catechol and amino groups, which was conducive to secondary reactions. In other words, PDA can enhance the binding force between scaffolds and drugs or growth factors through covalent or noncovalent bonding. The docking study of the PDA with SF scaffolds and E7 peptide was conducted using software to illustrate their interactive ability. The simulation results revealed the noncovalent bonding interaction forces between PDA to SF scaffolds and PDA to E7 peptide, for example, hydrogen bonds and electrostatic forces. The result also showed that the hydrogen bonds were primarily reflected in the catechol groups of PDA and the amino groups of SF or peptide. These new combinations increased the number of hydrophilic groups and facilitated cell adhesion. Besides, the interaction energy of the SF−PDA was −3.773 kcal/mol, higher than that of PDA-E7, which was about −3.047 kcal/mol. These results proved that the PDA had stronger interactions with SF scaffolds than PDA with E7, which might promote the release of E7 (Figure 2A). Moreover, since the reaction dose could reflect the binding quantity and primary amino groups of the corresponding molecules per unit area, QCM-D was employed to assess the real-time kinetics of PDA with SF as well as PDA with peptide (Figure 2B). According to the real-time frequency shift (Δf) of QCM-D, the frequency decreased when dopamine solutions were added in the QCM-D chambers, indicating PDA coating was gradually formed, which led to the increase in mass; 4 h later, Δf became stable. Subsequently, the injection of Trisbuffer solution was continued to wash away loosely bounded molecules. The quality of PDA did not change significantly, suggesting that the PDA coating formed on the sensor was stable and saturated. Accordingly, a total of 2600 ng/cm2 PDA was coated on the surface of the sensor. Next, the injections of SF solution and E7 solution were continued, separately. A total of about 1100 ng/cm2 SF and about 700 ng/cm2 E7 were coated on the PDA surface after the deposition of SF and E7 layers. Besides, large and rapid changes of Δf were observed on the PDA-coated surface, which suggested SF was easy to adsorb on the surface coated with PDA. Since the strong interaction between proteins and surface coated with PDA was attributed to the amino groups, it was also suggested that SF contained more active groups. This result also validated our docking analysis. The thermal decompositions of pure SF, pure PDA, and SF−PDA scaffolds in a nitrogen atmosphere were also conducted (Figure 2C). Nearly 7% weight loss attributed to water evaporation was detected in pure SF scaffolds around 30−125 °C. With further rise in temperature, at 400−450 °C, the weight residues started to decrease sharply due to the thermal degradation of SF scaffolds. After 600 °C, the residual weight was up to 50.5%. Similar thermal degradation behavior

Figure 3. Cell adhesion and morphology on the electrospun scaffolds. (A) BMSCs were seeded on the scaffolds for 12 h and then imaged by SEM (scale bars: 10 μm) and a confocal microscope (scale bars: 20 μm). (B) Cell viability on the electrospun scaffolds after seeding for 6 h and 12 h (n = 3, *p < 0.05 are differences between the indicated groups). G

DOI: 10.1021/acsami.8b22123 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 4. Cytotoxicity assay. (A) Live/dead staining and (B) live cell density of BMSCs grown on the electrospun scaffolds after 1 day of culture (scale bars: 250 μm); (C) cell proliferation of BMSCs grown on the electrospun scaffolds after 1, 3, 5, and 7 days of culture (n = 3, *p < 0.05 compared to the control group, #p < 0.05 compared to the SF group, +p