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Polylactic Acid Nanofiber Scaffold Decorated with Chitosan Islandlike Topography for Bone Tissue Engineering Ting Xu,†,§ Hongyang Yang,†,§ Dongzhi Yang,*,† and Zhong-Zhen Yu*,†,‡ †

State Key Laboratory of Organic-Inorganic Composites, College of Materials Science and Engineering and ‡Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China

ACS Appl. Mater. Interfaces 2017.9:21094-21104. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 04/30/19. For personal use only.

S Supporting Information *

ABSTRACT: In this work, a bicomponent scaffold with a core− shell and islandlike structure that combines the respective advantages of polylactic acid (PLA) and chitosan (CS) was prepared via electrospinning accompanied by automatic phase separation and crystallization. The objective of this research was to design nanosized topography with highly bioactive CS onto PLA electrospun fiber surface to improve the cell biocompatibility of the PLA fibrous membrane. The morphology, inner structure, surface composition, crystallinity, and thermodynamic analyses of nanofibers with various PLA/CS ratios were carried out, and the turning mechanism of a core−shell or islandlike topography structure was also speculated. The mineralization of hydroxyapatite and culture results of preosteoblast (MC3T3-E1) cells on the modified scaffolds indicate that the outer CS component and rough nanoscale topography on the surface of the nanofibers balanced the hydrophilicity and hydrophobicity of the fibers, enhanced their mineralization ability, and made them more beneficial for the attachment and growth of cells. Moreover, CS and “islandlike” protrusions on the fiber surface increased the alkaline phosphatase activity of the MC3T3-E1 cells seeded on the fibrous membrane and provided a more appropriate interface for cell adhesion and proliferation. These results illustrate that this kind of PLA/CS membrane has the potential in tissue engineering. More importantly, our study provides a new approach to designing PLA scaffolds, with combined topographic and bioactive modification effects at the interface between cells and materials, for biomedicine. KEYWORDS: electrospun, polylactic acid, chitosan, core−shell, island, tissue engineering

1. INTRODUCTION In recent years, electrospinning has become the most popular technology for the preparation of tissue-engineering scaffolds owing to its ability to produce nanofibrous scaffolds with high specific surface areas and porosities and with structural features that have a bionical morphology of the extracellular matrix (ECM).1,2 Compared with other scaffolds, electrospun nanofibers with isotropically aligned fibers have shown a superior ability to guide cell adhesion and migration and affect cell spreading and differentiation.3 Synthetic polyesters are widely used raw materials for electrospun tissue-engineering scaffolds. For example, polylactic acid (PLA),4,5 polycaprolactone,6,7 and poly(lactic-co-glycolic) acid8,9 have already been approved by the FDA (Food and Drug Administration) to be widely applied in the biomedical field because of their excellent degradability, mechanical properties, biocompatibility, and good designability. However, the cell affinity toward these polyester scaffolds is not satisfactory due to their higher hydrophobicity and fewer bioactive sites as compared to those of natural ECMs. Therefore, on the basis of the advantages of the electrospun fibrous membrane simulating the structure of natural ECMs, it © 2017 American Chemical Society

has been a challenge for polyester to achieve suitable cell adhesion and growth. Commonly, natural polymers10−15 with good bioactivity are preferred as modification components because they can provide cell recognition sites and render innate biological information guidance to cells. For example, electrospun chitosan (CS) scaffolds with good bioactivity have been broadly investigated in the tissue engineering field.16−18 However, their poor mechanical properties and rapid degradation rates limit their application.19,20 Recently, the blending nanofibers combining the advantages of PLA and CS have attracted the attention of researchers.21 For example, Nguyen22 and Wang et al.23 prepared PLA/CS core−shell nanofibers and Shalumon et al.24 reported that aligned PLA/CS blended nanofibers facilitated the aligned growth of human dermal cells. Au et al.25 focused on PLA/CS composite nanofibers with outstanding antibacterial activity for wound-healing applications, and Lee et al.26 successfully obtained a guided bone Received: January 24, 2017 Accepted: May 24, 2017 Published: May 24, 2017 21094

DOI: 10.1021/acsami.7b01176 ACS Appl. Mater. Interfaces 2017, 9, 21094−21104

Research Article

ACS Applied Materials & Interfaces Scheme 1. Preparation Process of CS Islanded-Structured Scaffolds

Our results demonstrated that this structure significantly enhanced the mineralization of HA and the attachment and growth of preosteoblasts (MC3T3-E1) on the scaffold’s surface, as we had expected. In addition, to demonstrate the mechanism of the formation of core−shell and islandlike structures on the surface of the fibers, the morphology, inner structure, surface composition, crystallinity, and thermodynamics of the nanofibers were studied in detail. The bone-bonding ability of the scaffolds was evaluated by investigating HA mineralization in a simulated body fluid (SBF). In addition, the behavior of MC3T3-E1 preosteoblast cells grown on the scaffolds was also investigated to explore their feasibility, used in tissue engineering.

regeneration membrane made of hydroxyapatite (HA) mineralized on a CS-coated electrospun PLA nanoweb. Various approaches have been demonstrated for the surface treatment of scaffolds, such as coating, layer-by-layer technologies,27 grafting via ray irradiation,28 and electrostatic interaction.29 For electrospun fibrous membranes, coaxial electrospinning is a common method for the preparation of multicomponent nanofibers with a desirable functional surface.30 It emphasizes a good match between the rheological properties of the inner and outer components for continuous and uniform core−shell nanofibers. Besides coaxial electrospinning technologies, single-nozzle electrospinning with a homogeneous or emulsion solution is another facile method to obtain core−shell structures. However, this method is favorable only for binary incompatible polymers in the same solvent system. Besides scaffold components, the topographic structure is also an important factor that affects the adhesion, growth, and cell differentiationand more importantly, improve protein adsorption.31 Theoretically, the simultaneous use of (i) outer CS bioactive components and (ii) a nanosized rough topography on the PLA nanofibers was more effective at finetuning the scaffold’s biocompatibility than the use of core−shell nanofibers with a smooth surface. Because of the automatic phase separation between two incompatible polymers during the electrospinning process, it is possible to switch the structure of the fibers from core−shell to “islandlike” with nanoscale protrusions. In our previous work, core−shell PEO/CS bicomponent nanofibers were first reported by a homogenous solution, and the formation mechanism was inferred to be the binodal phase separation of the ternary system in the fiber splitting process, accompanied by solvent evaporation.32 Similarly, for PLA/CS binary fibers, the size and shape of the CS island could be tuned by the solvent evaporation speed and the PLA crystallization process. A convenient and simple control of the electrospinning temperature served this purpose. Chitosan island-structured PLA nanofibers have the following essential advantages for improving the adaptation of cells to the scaffold surfaces: (1) a controllable balance of the surface hydrophilicity and hydrophobicity, (2) suitable roughness at the surface for cell attachment, and (3) cell recognition sites provided by the CS component, which provides innate biological information guidance to the cells. Furthermore, it is a facile and effective method that could be extended to other binary polymer scaffolds to improve the surface behavior of bulk materials.

2. EXPERIMENTAL SECTION 2.1. Materials. PLA with Mw of 150 000 g/mol was obtained from Shanghai Biodegradable Materials Technologies Co. Ltd. (Shanghai, China). Chitosan (DA 90%) was supplied by the Zhejiang Golden Shell Co. (Zhejiang, China). Trifluoroacetic acid (TFA) and all other chemicals for the preparation of the SBF were supplied by the Beijing Chemical Reagent Company (Beijing, China). All of the abovementioned reagents were used without further purification. The mouse preosteoblasts (MC3T3-E1) from calvarial bone were purchased from the Peking University Health Science Center (Beijing, China). The αminimum essential medium (α-MEM), ethylenediamine tetraacetic acid (EDTA), fluorescein isothiocyanate (FITC), and other reagents for the cell culture tests were supplied by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China) 2.2. Fabrication of Nanofibers by Electrospinning. The core− shell and islandlike structure of the nanofibers was regulated by the phase separation of PLA and CS in the PLA/CS homogenous solution by electrospinning. Our preparation process is shown in Scheme 1. The detailed process was as follows: 22 wt % CS and PLA were dissolved in TFA to form a homogeneous blending solution, using different CS/PLA weight ratios varying from 90:10 to 10:90. Pure CS and PLA solutions were used for comparison. The above solutions were taken in a 10 mL plastic syringe, which was fitted with a metal nozzle (with an inner diameter of 0.60 mm), and the collecting distance was set to be 20 cm. A 20 kV voltage and a spinning rate of 1.60 mL/h were applied. A perfect Taylor cone formed on the needle tip, and the well-electrospun PLA/CS nanofibers were collected onto the aluminum foil. Unless otherwise noted, the electrospinning temperature was controlled at 25 °C with relative humidity of 30%, collecting distance of 15 cm, and solution flow rate of 1.6 mL/h. For investigating the effect of temperature on the fiber surface morphology and the phase separation of PLA and CS, some electrospinning experiments were carried out at 35, 45, 50, and 60 °C for comparison. Finally, the nanofibers were dried at 40 °C in a vacuum oven for 24 h. Besides, there is another extraction process for removing the solvent and residual cross-linking agent glutaraldehyde before cell culturing on the fibrous membrane; the fibrous membrane was extracted in distilled 21095

DOI: 10.1021/acsami.7b01176 ACS Appl. Mater. Interfaces 2017, 9, 21094−21104

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Figure 1. (A−E) SEM images of electrospun PLA/CS nanofibers with different PLA/CS weight ratios at room temperature. (F) Histogram of the average diameter distribution of all electrospun nanofibers. (G−I) Fibers prepared at different temperatures using a PLA/CS 70:30 weight ratio; the island distance increased with an increase in temperature.The cross-linked PLA/CS nanofibers with weight ratios of (J) 80:20 and (K) 70:30 at 35 °C and etched with deionized water for 24 h; CS island remained on the fiber surface. (L) The non-cross-linked PLA/CS nanofibers with weight ratios of 70:30 at 35 °C and etched with deionized water for 24 h; nanofibers showed a smooth surface after removing the CS component. (M) Schematic of the islandlike structure. (N) Island size and distribution. With regard to the island height and vertical distance between islands, atomic force microscopy (AFM) analysis in the Supporting Information (Figure S1) also confirmed SEM results. 21096

DOI: 10.1021/acsami.7b01176 ACS Appl. Mater. Interfaces 2017, 9, 21094−21104

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For their observation with SEM, 3.7% formaldehyde−PBS solution and ethanol were used to fix and dehydrate the cells, respectively. For LSCM observations, 0.1% Triton X-100 was used to improve the permeability of the cell membrane. Afterward, cytoskeletal F-actin of cells was stained by a 50 μg/mL phalloidin labeled with tetramethylrhodamine isothiocyanate (TRITC) for 40 min and then rinsed with a PBS-removing redundant dye. Then, the cell nuclei were stained by Vectashield mounting medium with 4′,6-diamidino-2phenylindole (DAPI). The cell morphologies were visualized at 405 nm (DAPI) and 543 nm (TRITC) by an LSCM.37 2.9. Alkaline Phosphatase (ALP) Activity Test and Osteocalcin (OCN) Expression. ALP activity of the cells was tested after seeding for 3, 7, 14, and 21 days according to the literature.32 Specifically, 400 μL of a p-nitrophenyl phosphate and disodium salt solution was added into each well and allowed to react for 1 h. Thereafter, 200 μL of a 2 M NaOH solution was transferred into each well using a pipettor. Finally, 100 μL of the reaction product was transferred into a blank 96-well microplate reader and then the absorbance was tested at 450 nm. For the OCN expression, the cell lysate was detected by an ELISA kit at 450 nm after culturing the cells for 7 and 14 days.

water for 48 h, and the medium was refreshed every 4−6 h until the pH was close to neutral. 2.3. Fluorescence Labeling of CS. To prove that CS mainly existed in the shell of the nanofibers, the nanowebs (1 cm × 1 cm) were immersed in a 0.1 wt % methanol solution of fluorescein dye FITC for 8 h in the dark (the volume ratio of methanol and deionized water was 10:4, pH 9). Then, the membranes were rinsed with ethanol and distilled water to remove the unreacted FITC, dried, and analyzed by laser scanning confocal microscopy (LSCM).33,34 2.4. Mineralization of the Nanowebs with 10× SBF. Mineralization of the PLA/CS nanowebs was carried out in 10× SBF after cross-linking with glutaraldehyde vapor (50 wt % aqueous solution) for 3 h at 25 °C. A saturated solution of 10× SBF was prepared with 5.49 g of NaCl, 0.36 g of NaHCO3, 0.29 g of CaCl2, 0.36 g of Na2HPO4·12H2O, and 100 mL of deionized water. The PLA/CS nanowebs (3 cm × 3 cm) were immersed in the above 10× SBF solution for 3−24 h at 37 °C with mild stirring. The SBF solution was refreshed at every 3 h interval. When taken out from the 10× SBF solution, the samples were treated gently with deionized water and then vacuum drying was carried out at 40 °C for 24 h.35,36 2.5. Characterization. The surface morphology of the prepared nanofibers was characterized using a scanning electron microscope (SEM, S-4700; Hitachi Company, Japan). The inner structure of the nanofibers was observed by a transmission electron microscope (TEM, H-800; Hitachi Company, Japan). X-ray photoelectron spectroscopy (XPS, Escalab 250; Thermo Fisher Scientific Corporation) was chosen to analyze the composition of the outer layer of the nanofibers. The crystalline behavior and thermodynamic behavior of the fibers were respectively investigated using a wide-angle X-ray diffraction (XRD) analyzer (D8 ADVANCE; Bruker AXS Gmbh, Germany) and a differential scanning calorimeter (DSC; Q100 TA Instruments), under a rate of 10 min−1 from 30 to 100 °C. The elemental and content analyses of the mineral HA crystals deposited on the PLA/CS nanofibers were performed by an energy-dispersive spectrometer (EDS) attached to the SEM system. The surface hydrophilicity of the PLA/CS nanofibrous membranes was evaluated by a contact angle goniometer (DSA-30; Kruss, Germany); five sites on the same membrane were detected. The fluorescence intensity of the FITClabeled nanofibers and the growth and adhesion of the cells were observed by a laser scanning confocal microscope (LSCM, Leica SP5; Leica Microsystems Wetzlar GmbH, Germany). An exciton of the fluorophore with a 488 nm Ar−Kr laser was used to characterize the FITC-labeled nanofibers. 2.6. Cell Culture. Mouse preosteoblasts MC3T3-E1 were cultured in the α-MEM with 10% fetal bovine serum (FBS), 1% penicillin/ streptomycin, 3 mM β-glycerophosphate, and 10 mg/mL ascorbic acid at 37 °C and in 5% CO2. The medium was refreshed every 24 h. The cells were trypsinized by 0.25% trypsin with 1 mM EDTA when they reached 80% confluence. 2.7. Cell Proliferation. The samples were sterilized in highly compressed steam for 20 min. After that, the MC3T3-E1 cells were seeded on the membrane with a density of 3 × 103 cells per well in 96well plates. At 1, 2, 3, 7, and 14 days after culturing, the cell viability was detected with CCK-8 at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader (Multiscan MK3; Labsystem Co., Finland). For comparison, α-MEM with 10% FBS and the same medium with 1% phenol were chosen as the negative control and positive control, respectively. The number of cells adhered on the nanofiber membrane was calculated according to the functional relation between the absorbance and the number of cells.3 2.8. Cell Spreading Morphology Observation by SEM and LSCM. To observe the attachment ability of the cells and their shapes on the different scaffolds, all scaffolds were separately fixed on glass coverslips, sterilized in a 70% ethanol solution overnight, then rinsed with a sterile phosphate buffer saline (PBS) solution three times, and finally placed into 24-well plates pretreated with a poly(2-hydroxyethyl methacrylate) solution to avoid the attachment of the cells to the plates. Approximately, 3 × 103 trypsinized cells/mL were seeded on each sample and cultured for 1, 2, 3, 7, and 14 days.

3. RESULTS AND DISCUSSION 3.1. Structure and Morphology of Nanofibers. In this section, we focused on the effects of the PLA and CS component ratios and the electrospinning temperature on the inner and outer phase separation structure of the fibers. The surface morphologies of the nanofibers prepared varying the PLA/CS weight ratios from 0 to 100% at the same temperature and varying the temperature with a fixed PLA/CS ratio of 70:30 are displayed in Figure 1A−E. Figure 1G−I also show the morphology of the nanofibers prepared at different temperatures but with a fixed PLA/CS ratio of 70:30. The results indicate that pure PLA fibers with an average diameter of about 1 μm showed a smooth and uniform surface morphologies without any beads. The PLA/CS fiber diameters decreased as the CS content increased, whereas the polydispersity increased. Chitosan fibers with 200 nm diameter were uneven and fragile. In Figures 2B,C and S2A−C, TEM micrographs display the clear interface boundary between the core and shell structures

Figure 2. TEM images of electrospun PLA/CS. with the weight ratio of (A) 100:0, (B) 70:30, (C)10:90 and (D) 0:100; (E) 70:30 of PLA/ CS at 35 °C and etched by dichloromethane. Compared to pure PLA or CS, all PLA/CS composite fibers display core−shell structures prepared at room temperature. A sharp contrast of the bright and dark regions in the TEM image displays the morphology of a hollow interior but an external continuous cellular structure, proving the distribution of binary polymer PLA and CS in the islandlike fibers. 21097

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Figure 3. XPS spectra of C 1s region of (A) pure PLA, (B) pure CS and (C) PLA/CS (70:30) electrospun nanofibrous membranes. (D) the survey scan of (a) PLA, (b) CS, (c) PLA/CS (70:30) and (d) PLA/CS (70:30) nanofibrous films after etching in deionized water for 24 h.

of the electrospun PLA/CS nanofibers, with varying CS ratios, owing to the phase separation of PLA and CS accompanied by solvent evaporation. Figure 1G−I show the morphologies of electrospun PLA/CS (70:30) fibers prepared at different temperatures. Compared to the smooth nanofibers obtained at 25 °C, those nanofibers became rough and many small bumps with an islandlike structures appeared on their outer surfaces. To confirm the major composition of the shell, the surface morphologies of the fibers before and after crosslinking, and etched by deionized water was characterized by SEM images. As shown in Figure 1K,L, for cross-linked sample, the “island” structure on the outer surface still existed, while that disappeared for non-cross-linked sample. This phenomenon illustrates that the islandlike CS structure appeared because CS is soluble in aqueous solutions but PLA is not. To further illustrate the composition of the “sea” and island segments, electrospun PLA/CS (70:30) nanofibers prepared at 35 °C were collected on a copper net and then etched with dichloromethane. The sharp contrast between the bright and dark regions in the TEM image (Figure 2E) displays the morphology of a hollow interior but an external continuous “cellular” structure. Combined with the TEM micrograph (Figure 2B) of the rinsed fibers with a core−shell and an islandlike structure, the phenomenon further demonstrated that the sea was made of PLA because dichloromethane dissolves PLA but not CS. Meanwhile, with the exception of the PLA/CS 70:30 weight ratio, electrospun PLA/CS 80:20 nanofibers at 35 °C also presented the similar islandlike structure, as shown in Figure 1J. The above speculation could also be further proved by the XPS analysis. We speculated that the phase separation of PLA and CS occurred during the solvent evaporation step of

the electrospinning process. One possible reason for the immigration of the CS phase to the surface of the fibers is that CS with positively charged groups prefers to move out under an electrical field during electrospinning. Some eccentricity was found in Figure 2C, presumed to be caused by a larger number of positively charged CS units, leading to some instability of the composite solution and strong whipping during electrospinning. These results were easily understood by the increase in the viscosity of the PLA/CS solution, with the evaporation of the solvent, allowing PLA to block the aggregation of the CS molecules. In addition, the repulsive electrical force of intermolecular interactions also hindered CS from gathering. Therefore, the PLA sea and CS island structure was formed. Furthermore, the size and distribution of CS islands could be easily regularized by controlling electrospinning temperature. As shown in Figure 1M,N, an increase of the electrospinning temperature accelerated the solvent evaporation rate and the number of CS molecules that migrated to the surface of the fiber was reduced. This caused the periodic distance of the islands to increase and the size of the islands to decrease. To optimize the fiber morphology and film strength, we selected the PLA/CS 70:30 ratio in the following experiment. By increasing the temperature from 35 to 60 °C, the periodic distances of islands obviously increased from 400 to 750 nm and the island size decreased from 200 to 140 nm. In addition, the topographical analysis of the fiber surface (AFM) was also used to characterize the nanofibers (Figure S1). The aligned PLA/CS islandlike fibers were collected in the gap (3 cm) between the two pieces of magnetite at 35 °C, and the island height and distance between the islands were recorded by AFM analysis software. As shown in Figure 1N, the island height is 21098

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Figure 4. (A) XRD and (B) DSC curves of PLA/CS nanofibers with different PLA/CS blending ratios: (a) raw 100:0; (b) PLA, (c) 70:30, (d) 50:50, (e) 30:70, and (f) 0:100. (C)XRD curves and (D) DSC curves of PLA/CS (70:30) nanofibers prepared at different temperatures: (a) 25 °C, (b) 45 °C, and (c) 60 °C.

3.3. LSCM Analysis of FITC-Labeled Nanofibers. To further confirm that the main component in the shell of the PLA/CS nanofibers was CS, FITC-labeled nanofibers were observed by LSCM. The NCS groups of FITC could interact with the −NH2 groups of CS. As shown in Figure S3, there was no fluorescence in pure PLA nanofibers, whereas green fluorescence was visible on the surface of the PLA/CS nanofibers, indicating some component on the surface of the PLA/CS nanofiber reacted with FITC, and it is a supporting evidence for CS mainly present in the shell of the nanofibers. 3.4. Crystallinity and Thermodynamic Analysis. The XRD and DSC curves of PLA/CS nanofibers, synthesized using different blending ratios and electrospinning temperatures, demonstrated the changes in the crystallinity and thermodynamics of PLA. As shown in Figure 4A, raw PLA showed strong diffraction peaks at 16.7 and 19.0°, corresponding to the (200)/ (110) and (203) crystal planes, and relatively weak peaks at 14.9 and 22.3°. No acute diffraction peaks were observed in the PLA nanofibers, contrary to those in the raw PLA particles, the diffraction intensity of the blending nanofibers presented an obvious downtrend as the CS content increased. The DSC curve of CS is shown in Figure 4B. CS is a crystalline polymer, implying it could not be melted, so its DSC curve showed a smooth line. However, the glass-transition (Tg), coldcrystallization (Tc), and melting (Tm) temperatures of PLA were clearly visible on the DSC curve of the PLA/CS nanofibers. The Tg of PLA presented a slight tendency to increase in proportion to CS. This could be interpreted by the partial miscibility of CS and PLA. In fact, the entanglement between the PLA and CS molecular chains in the blending

about 50 nm, and the vertical distance between islands is about 700 nm. This phenomenon will be explained in “3.5 Turing Mechanism of the Island and Core−Shell Structure”. 3.2. Chemical Composition Analysis of the Shell Layer. To prove that the major component of the outer layer was CS, an XPS analysis was carried out. Figure 3A−C show the XPS C1s spectra of PLA, CS, and PLA/CS (70:30) core−shell electrospun fibers, respectively. As shown in Figure 3A, the C1s signals attributed to the C−C, C−O, and CO bonds of PLA were located at 284.5, 285.2, and 298.5 eV, respectively. Compared with pure PLA nanofibers, an obvious difference could be found in the PLA/CS (70:30) nanofibers (Figure 3C), not only for the C1s signals of the C−C, C−O, and CO bonds, but also for the C−N bond at 288.6 eV, which belongs to CS. For the elemental content analysis (C, O, and N), the N atomic percentage in the PLA/CS (70:30) sample was 5.1%, which was close to that of 5.9% observed for the pure CS sample. This illustrates that the major component on the outer surface of the PLA/CS nanofibers is CS. In comparison with the C1s spectrum of CS in Figure 3B, the shift of the C−N bond in the PLA/CS fibers from 287.6 to 288.6 eV might be caused by the interaction between the PLA and CS molecules. By comparing Figure 3D(c,d), a clear N1s signal disappeared after the fibers were rinsed with deionized water. Hence, the results further confirm that the main component of the core was PLA, whereas CS mostly existed in the shell layer. The reason for this behavior might be that the positively charged amino groups of CS tended to move out due to electrostatic repulsion. 21099

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As shown in the XRD patterns, the characteristic diffraction peaks of the crystals, such as the (002), (210), (300), and (203) lattice planes at around 2θ = 26.0, 27.6, 31.7, and 45.5°, respectively, and the Ca/P = 1.63 tested by EDS, wellcorresponded with the HA crystals. Conversely, only a few mineral particles were deposited on the pure PLA fibrous material after a 24 h incubation time. Previous studies39 have concluded that enhanced hydrophilic properties, which could be introduced by using certain functional groups, such as −OH and −NH2, could accelerate the deposition of calcium phosphate. This could enrich the amount of Ca2+ on the matrix surface and lead to the local supersaturation of Ca2+ and crystal nucleation. Therefore, our results confirmed that CS, which has abundant −OH and −NH2 groups, played a key role in the formation of HA on the surface of PLA/CS scaffolds. The porosity of the electrospun fibrous membrane was measured using the following equations

system blocked the free motion of the PLA chains, which was accompanied by the migration of the CS molecular chains to the surface of the nanofiber and the formation process of the core−shell-structured nanofibers. In addition, compared with the pure PLA nanofibers, an obvious cold-crystallization process appeared in the range of 100−160 °C for all the PLA/CS blended systems, whereas double melting peaks of PLA appeared at 160−180 °C, especially for the PLA/CS (30:70) nanofibers, as shown in Figure 4B(d,e). These results confirmed that a significant amount of CS molecular chains interfered with the crystallization of PLA during the electrospinning process. As shown in Figure 1, the electrospinning temperature also strongly affected the fiber morphology. To corroborate our interpretation, the effect of temperature on the crystallization and thermodynamic behavior of the blended nanofibers was investigated. The XRD pattern of PLA/CS (70:30) in Figure 4C(c) shows that there was no significant difference in the diffraction peaks at a higher electrospinning temperature while the melting temperature (Tm) of PLA obviously decreased. This is because of the inhibition of crystallization by the rapid solvent evaporation at higher temperatures. The thermal properties of pure PLA and PLA/ CS nanofibrous membranes based on the DSC curves (Figure 4B,D) are summarized in Table S1. 3.5. Turning Mechanism of the Island and Core−Shell Structure. To explain the formation mechanism of the core− shell and island structures, the ternary phase diagram of the binary polymer−solvent system to track the change of the component proportion during the solvent evaporation. As shown in Figure S4, with the solvent evaporation and cooling, electrospinning solution gradually turned to be unstable or metastable leading to nucleation and growth (NG, binodal phase separation) or spinodal decomposition (SD, spinodal phase separation) and thus phase separation occurred. In more detail, our previous work already explained the core−shell structure in the PEO/CS electrospinning system by this theory.32 In addition, with the solvent evaporation, the charged groups of the cationic CS molecular chains preferred to immigrate out under electrostatic repulsion. There was an interesting phenomenon regarding why the island structure appeared under a higher electrospinning temperature, not a core−shell structure. It was easy to understand that the evaporation of solvent was quicker under a higher temperature and there was not enough time to migrate to the surface of electrospun jets for some CS molecular chains, so that there was not enough CS component to cover the whole fibrous shell, and the intermittent islands structure appeared. 3.6. Mineralization Analysis. The mineralization behavior of the PLA/CS fibrous scaffold in a 10× SBF solution for varying time periods (3, 6, 9, 12, and 24 h) was characterized by SEM, EDS, XRD, and thermogravimetric analysis. As shown in Figure S5, and in comparison to that of a pure PLA fibrous film, a significant increase in the number and size of the mineral crystals was observed on the surface of the PLA/CS (70:30) sample as the mineralization time increased. After mineralization for 9 h, a thin mineral layer formed on the surface of the nanofibers. After 24 h, the porous structure of the fibrous scaffold had been fully covered by large mineral crystals and a highly roughened structural layer. The amount of deposited minerals gradually increased with the incubation time, and the weight percentages by TGA analysis were 22.2, 41.1, 61.9, and 68.2%, as shown in Figure S6.

apparent density(g/cm 3) = mat mass (g)/mat volume (cm 3)

Porosity = [1 − PLA mat apparent density(g/cm 3) /bulk density of PLA(g/cm 3)] × 100%

For pure PLA mat, the porosity is 85 ± 5%, whereas for PLA/ CS islandlike mat, it is 93 ± 4%. Typical stress−strain curves of the PLA and PLA/CS fibrous membranes showed the different initial modulus of the membrane and tensile strength (Figure S7), and the tensile strength and break elongation (%) of the cross-linked fibrous membrane (PLA/CS 70:30) are 2.0 MPa and 20%, respectively. 3.7. Adhesion and Growth of MC3T3-E1 Cells. The adhesion and proliferation of cells on the surface of biomaterials are key steps in tissue engineering. Figure 5 presents the CKK-8

Figure 5. CCK-8 assay of MC3T3-E1 cells in the extract of pure PLA, PLA/CS 70:30, and mineralized PLA/CS scaffolds after culturing for 1, 2, 3, 7, and 14 days (*p > 0.05).

test results, which revealed the proliferation of MC3T3-E1 cells seeded on different scaffolds for 1, 2, 3, 7, and 14 days. It can be seen that, in comparison with the negative control, no statistically significant difference was detected in MC3T3-E1 cell numbers after culturing for 3−14 days in the presence of PLA/CS membrane. In addition, the viability of the cells was above 95%. For pure PLA and PLA/CS with island structure scaffold, statistically significant differences (p < 0.05) were observed after seeding 2 days. These results indicate that the CS islandlike structure was beneficial for cell proliferation. In addition, visible increases of MC3T3-E1 cell numbers with the 21100

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Figure 6. LSCM micrographs (400×) of cells attached and grown on different scaffolds, stained with DAPI (the images in the first, second, and third rows corresponded to 1, 2, and 3 day, respectively): (A) PLA (A1, A2, A3); (B) PLA/CS 70:30 (B1, B2, B3) with a core−shell structure; and (C) PLA/CS 70:30 (C1, C2, C3) with an islandlike structure. Visible increases of MC3T3-E1 cell numbers with the culture time are shown (Figure S8). After 3 days of culturing, the number of cells on the island PLA/CS membrane was significantly higher than that on the other samples.

Figure 7. SEM and LSCM micrographs (400×) of the cells grown and attached for 48 h on (A) pure PLA(A1, A2); (B) PLA/CS 70:30 with a core−shell structure (B1, B2), and (C) PLA/CS 70:30 with an island structure (C1, C2), stained with DAPI and TRITC-phalloidin.

culture time are shown in Figures 6 and S8. After 3 days of culturing, the cell numbers on the island PLA/CS membrane

were significantly higher than those on the other samples. This indicates that the combination of CS and rough nanosized 21101

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Figure 8. (a) ALP activity of MC3T3-E1 cells grown on the nanofibrous membranes cultured for 3, 7, 14, and 21 days. (b) OCN expression of MC3T3-E1 cells on membranes cultured for 7 and 14 days; * denotes a significant difference as compared to control (*P < 0.05).

3.8. ALP Activity Test and OCN Expression of MC3T3E1 on the Membrane. To evaluate the differentiation behavior of MC3T3-E1 cells on the membrane with different surface components and protrusion structures, ALP activity and OCN expression measurements are essential. ALP is an early marker for osteoblast differentiation, and OCN is a typical marker of biomineralization of bone and mature osteoblasts. As shown in Figure 8, in comparison to those on the PLA scaffold, the ALP activities of cells cultured on the islandlike and core− shell PLA/CS scaffolds were both enhanced. In addition, at the day 7 and day 14 time points, the cells on the surface of the fibers with rich CS showed a significantly higher expression of OCN, especially on the islandlike PLA/CS scaffold. Early studies have noted that changes in the shape of cells can affect the differentiation and that a greater cytoskeletal tension of the cells tends to enhance their osteogenesis ability.39 Additionally, the gene expression was investigated including ALP, osteopontin (OPN), and OCN of MC3T3-E1 cells during the 21 days. As shown in Figure 9, compared to that on the pure PLA membrane, there was a significantly higher ALP, OPN, and OCN gene expression on the PLA/CS membrane after 7 and 21 days (*p < 0.05). After 7 days culturing, the ALP gene expression increased slowly but the OCN expression significantly increased. Compared two kinds of PLA/CS nanofiber surfaces, core−shell and islandlike structure, at early time 7 days, PLA/CS islandlike membrane showed a higher ALP and OPN gene expression, while higher OCN gene expression at 21 days. All of the data supported our LSCM results of cells spread on scaffolds (Figure 7). The cells grown on islandlike scaffolds showed elongated morphologies and enhanced cytoskeletal tension.

morphologies played a positive role on the attachment and growth of the cells. To more visually observe the spreading behavior of the MC3T3-E1 cells on the membrane, the cells were stained for 48 h with DAPI, for the nuclei of the cells, and TRITC-phalloidin for the cytoskeletal actin, to reflect the difference of cell attachment and cytoskeleton shape on pure PLA and PLA/CS with a core−shell or island structure. As shown in Figure 7, the number of adheredcells and the spreading morphology on PLA/CS membrane of both core− shell and island structures were obviously better than those on the pure PLA sample, especially for island PLA/CS, indicating that this nano-topology surface was more favorable for the spread of cells. Also, SEM images with a larger magnification provided a more clear characterization of cellular spreading after culturing 96 h, as shown in Figure S9. In a dynamic environment, cells respond to external stimuli by changing their function or by reorganizing their cytoskeleton. Cells interact with the bioactive sites on the material surface through receptors on their surface, such as integrin. The interaction between biomaterials and cells is essential to enhance the cell adhesion and growth on the material. Cell adhesion is the basis, as it will affect cell proliferation, differentiation, and other functions. Cell adhesion on the material surface is divided into four steps: cell attachment, spreading, organization of the actin cytoskeleton, and formation of focal adhesions. From a biomaterials viewpoint, the adhesion and growth of cells on the surface of a material are strongly influenced by the surface properties of the biomaterial, such as the balance of hydrophilicity and hydrophobicity, surface roughness, and whether there are bioactive sites for anchoring the cells. In this article, to enhance the bioactivity of cells on the surface of scaffolds, CS was used to modify the surface of the PLA scaffold, so as to modify the abovementioned three parameters. For example, we provided active sites for integrin and cell adhesive proteins and changed the scaffold surface from hydrophobic to hydrophilic, as many studies have demonstrated that the aforementioned cell behaviors, that is, adhesion and growth, are more favorable on moderately hydrophilic interfaces than on hydrophobic ones.38 On the other hand, the roughness of the modified nanoscale surface and the surface topology of the scaffold were also significantly changed by the phase separation between PLA and CS. In general, the cells orient and migrate along the protrusions or grooves on the surface of a material. This phenomenon is called the contact induction of cell culture.

4. CONCLUSIONS In this study, PLA/CS composite scaffolds with core−shell and islandlike surface structures were prepared via electrospinning, accompanied with an automatic phase separation controlled by the evaporation of the solvent. The formation mechanism of the above structures was discussed using the ternary phase diagram of polymer solutions. Surface CS-coated PLA scaffolds were used to investigate the combining effects of bioactive molecules and topography on the adhesion and growth behavior of preosteoblast MC3T3-E1 cells. The results showed that PLA scaffolds with CS island-nanotopology surfaces were more favorable for the spread of cells. In addition, the CS component on the fibrous surface accelerated the mineralization of HA, as well as endowed the scaffold with a better ability 21102

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structure; SEM images, XRD spectra, EDS and TGA curves of mineralization of nanofibrous membranes; LSCM micrographs of the cells cultured on samples; SEM images of the cells grown and attached for 12 and 96 h on different samples (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel/Fax: +86-10-64428582 (D.Z.Y.). *E-mail: [email protected] (Z.Z.Y.). ORCID

Dongzhi Yang: 0000-0003-2592-7833 Zhong-Zhen Yu: 0000-0001-8357-3362 Author Contributions §

T.X. and H.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The author would like to thank the National Natural Science Foundation of China (51273015) for its financial support. (1) Kim, T. G.; Shin, H.; Lim, D. W. Biomimetic Scaffolds for Tissue Engineering. Adv. Funct. Mater. 2012, 22, 2446−2468. (2) Yao, Q. Q.; Cosme, J. G.; Xu, T.; Miszuk, J. M.; Picciani, P. H.; Fong, H.; Sun, H. L. Three Dimensional Electrospun PCL/PLA Blend Nanofibrous Scaffolds with Significantly Improved Stem Cells Osteogenic Differentiation and Cranial Bone Formation. Biomaterials 2017, 115, 115−127. (3) Li, N.; Chen, G.; Liu, J.; Xia, Y.; Chen, H. B.; Tang, H.; Zhang, F. M.; Gu, N. Effect of Surface Topography and Bioactive Properties on Early Adhesion and Growth Behavior of Mouse Preosteoblast MC3T3-E1 Cells. ACS Appl. Mater. Interfaces 2014, 6, 17134−17143. (4) Valente, T. M. A.; Silva, D. M.; Gones, P. S.; Fernandes, M. H.; Saneos, J. D.; Sencadas, V. Effect of Sterization Methods on Electrospun Poly(Lactic Acid) (PLA) Fiber Alignment for Biomedical Applications. ACS Appl. Mater. Interfaces 2016, 8, 3241−3249. (5) Li, J. S.; Chen, Y.; Mak, A. F. T.; Tuan, R. S.; Li, L.; Li, Y. A OneStep Method to Fabricate PLLA Scaffolds with Deposition of Bioactivehy Droxyapatite and Collagen Using Ice-Based Microporogens. Acta Biomater. 2010, 6, 2013−2019. (6) Xu, T.; Miszuk, J. M.; Zhao, Y.; Sun, H. L.; Fong, H. Electrospun Polycaprolactone 3D Nanofibrous Scaffold with Interconnected and Hierarchically Structured Pores for Bone Tissue Engineering. Adv. Healthcare Mater. 2015, 4, 2238−2246. (7) Shalumon, K. T.; Anulekha, K. H.; Girish, C. M.; Prasanth, R.; Nair, S. V.; Jayakumar, R. Single Step Electrospinning of Chitosan/ Poly(Caprolactone) Nanofibers using Formic acid/Acetone Solvent Mixture. Carbohydrate Polymers 2010, 80, 413−419. (8) Kumbar, S. G.; Nukavarapu, S. P.; James, R.; Nair, L. S.; Laurencin, C. T. Electrospun Poly (Lactic Acid-co-Glycolic Acid) Scaffolds for Skin Tissue Engineering. Biomaterials 2008, 29, 4100− 4107. (9) Zhou, P. Y.; Cheng, X. S.; Xia, Y.; Wang, P. F.; Zou, K. D.; Xu, S. G.; Du, J. Z. Organic/Inorganic Composite Membranes Based on Poly(L-Lactic-co-Glycolic Acid) and Mesoporous Silica for Effective Bone Tissue Engineering. ACS Appl. Mater. Interfaces 2014, 6, 20895− 20903. (10) Zhou, T.; Wang, N. P.; Xue, Y.; Ding, T. T.; Liu, X.; Mo, X. M.; Sun, J. Development of Biomimetic Tilapia Collagen Nanofibers for Skin Regeneration Through Inducing Keratinocytes Differentiation and Collagen Synthesis of Dermal Fibroblasts. ACS Appl. Mater. Interfaces 2015, 7, 3253−3262.

Figure 9. Gene expression of bone-associated mRNAs by MC3T3-E1 grown on PLA/CS electrospun fibrous scaffolds. MC3T3-E1 initial differentiation (day 0) and after an additional culturing for 7 and 21 days. Expressions of (A) ALP, (B) OPN, and (C) OCN mRNAs were tested by qRT-PCR. The mRNA level was normalized against glyceraldehyde 3-phosphate dehydrogenase and are presented as fold changes relative to PLA at day 0. Four independent experiments were carried for gene expressions (n = 4). Statistical analysis: p < 0.05.

to form bone, as proved by the ALP activity evaluation and OCN expression of the cells. There are reasons to believe that our method would have a practical application for improving the interface of tissue-engineering scaffolds, and this method may be popularized among other binary polymer blends.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01176. TEM images of electrospun PLA/CS; LSCM images of electrospun PLA and PLA/CS; AFM analysis of the fiber surface; mechanical properties of the fibrous membrane; DSC result of electrospun PLA/CS; schematic diagram of the formation mechanism of core−shell or island 21103

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