Enhanced osteogenesis of bone morphology protein-2 in 2-N,6-O-sulfated chitosan immobilized PLGA scaffolds

Colloids and Surfaces B: Biointerfaces 122 (2014) 359–367

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Enhanced osteogenesis of bone morphology protein-2 in 2-N,6-O-sulfated chitosan immobilized PLGA scaffolds Xiangjun Kong c , Jing Wang c,∗ , Lingyan Cao c , Yuanman Yu c , Changsheng Liu a,b,c,∗ a

The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China b c

a r t i c l e

i n f o

Article history: Received 5 April 2014 Received in revised form 23 June 2014 Accepted 10 July 2014 Available online 18 July 2014 Keywords: Scaffold Surface modification Sulfated chitosan Bone morphology protein-2 Osteogenesis

a b s t r a c t By using 2-N,6-O-sulfated chitosan (26SCS) immobilized poly(lactide-co-glycolide) (PLGA) scaffolds, our system achieved controlled release and improved bioactivity of recombinant human bone morphology protein-2 (rhBMP-2). Initially aminolyzed by ethylenediamine, PLGA scaffolds surface was immobilized with 26SCS via electrostatic assembly. Upon the presence of 26SCS, the system displayed improved rhBMP-2 adsorption and prolonged release process in vitro due to the high affinity of rhBMP-2 with 26SCS. On the other hand, because of incorporation of 26SCS, the system appeared to be more hydrophilic and provided a better environment for cells attachment. Moreover, 26SCS enhanced the binding efficiency between rhBMP-2 and its receptors as well as alkaline phosphatase activity. Our study highlights 26SCS immobilized PLGA scaffolds may be excellent candidates for use in bone tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Biodegradable polymeric scaffolds for bone tissue engineering have received much attention since they provided a temporal and spatial environment for cellular growth and bone repair [1–3]. Especially, poly(␣-hydroxyester)s-type biodegradable polymers, including poly (l-lactide) (PLLA) [4], polyglycolide (PGA) [5] and its copolymer poly(lactide-co-glycolide) (PLGA) [6], possess adjustable degradation rate, good biocompatibility and suitable mechanical property. Furthermore, they are approved by the Food and Drug Administration (FDA). However, lacking of cell anchorage sites, poor hydrophilicity and lower surface energy impede the cell attachment and penetration on these polymeric scaffolds [7,8]. On the other hand, growth factors are often used in polymeric scaffolds to transmit signals and modulate cellular activities [9]. Bone morphology protein-2 (BMP-2), belonging to the transforming growth factor-␤ (TGF-␤) superfamily, plays critical roles in osteogenesis and bone metabolism by facilitating cellular proliferation and differentiation of a variety of cell types including osteoblasts and chondrocytes [10]. Nevertheless, BMP-2 is easy to

∗ Corresponding authors at: No. 130, Meilong Road, Shanghai 200237, PR China. Tel.: +86 21 64251308; fax: +86 21 64251358. E-mail address: [email protected] (J. Wang). http://dx.doi.org/10.1016/j.colsurfb.2014.07.012 0927-7765/© 2014 Elsevier B.V. All rights reserved.

lose bioactivity over a short half-life and do not always exhibit the required efficacy in bone regeneration under physiological conditions in vivo [11]. Furthermore, a high dosage of BMP-2 may be a high cost and accompanied with contingent risk, such as osteoarthritis and sclerosteosis [12]. Consequently, improving the cellular affinity as well as endowing ameliorative reservoir of bioactive growth factors as a controlled delivery system becomes increasingly necessary. Within past decades, considerable researchers have attempted to mimic the natural extracellular matrix by immobilizing naturally derived biomolecules onto polymer scaffolds [7,13,14], including the representative heparin functionalized biodegradable scaffolds for local and sustained release of BMP-2 [15–17]. Heparin, a highly anionic linear polysaccharide, prolongs the half-life of BMP-2 and improves its bioactivity by specific interactions with BMP-2:strong bind between heparin sulfated groups and the BMP-2 residues [18]. Despite heparin/BMP-2 promoted bone regeneration, it is cautious to use heparin for the possible delayed bone healing in shortterm use and increased risk of osteoporosis in long term treatment [19,20]. These drawbacks might be critical obstructions for heparin in the clinical application. As a result, it is rather important to seek for an alternative which can effectively improve BMP-2 bioavailability. To satisfy current needs, sulfated chitosan as a kind of heparin-like polysaccharide, especially 2-N,6-O sulfated chitosan (26SCS), owning the similar structure of heparin and achieving

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higher degree of sulfation than any natural sulfated polysaccharides, has attracted great interests in recent years. Furthermore, our previous study demonstrated that 26SCS is of low cytotoxicity and is a potent synergistic factor of BMP-2 for local bone regeneration [21,22]. Functional biomolecules, such as 26SCS, can be immobilized onto the surface of scaffolds by physical adsorption, electrostatic assembly or covalent binding [23,24]. The combined biomolecules by physical adsorption are simple but unstable. Although covalent binding is the most stable method, the process is relatively complex. Compared with above tactics, electrostatic assembly is a facial, convenient and comparatively stable method to immobilize biomolecules and meet the application demands. Based on the above rationale, this work developed a 26SCSimmobilized porous PLGA scaffolds by electrostatic assembly. Briefly, upon initial aminolysis, the PLGA scaffold surface was immobilized with 26SCS via electrostatic interaction between negatively charged sulfated groups of 26SCS and positively charged amine amino residues on PLGA scaffolds. Physicochemical properties in terms of surface topography, hydrophilia and composition were investigated. RhBMP-2 binding efficiency and release profiles were examined. Furthermore, in vitro cellular affinity including cell attachment, proliferation and osteogenic differentiation were inspected with C2C12 cell line. We rationalized that 26SCSimmobilized PLGA scaffolds will not only endow more desirable cellular response, but also enhanced osteoinductive activity due to the synergistic effect between rhBMP-2 and 26SCS. 2. Materials and methods

PLGA scaffolds were immersed in ethylenediamine (ED) at various concentrations (0.05, 0.1, 0.2 mol/L) for the desired time period under room temperature. Samples were washed in ice cold water for a further time. Then the aminated scaffolds were soaked in 10 mg/mL SCS solution for 2 h and washed at least 3 times to remove unreacted SCS remnant and dried in a vacuum oven at 37 ◦ C for 24 h. The PLGA scaffolds without modification were set as a negative control. The resulting scaffolds were stored in a desiccator until further use and named as PLGA, S-PLGA0.05 , S-PLGA0.1 , S-PLGA0.2 respectively (0.05, 0.1, 0.2 represented the concentration of ED). 2.4. Morphology characterization 2.4.1. Scanning electron microscopy The surface morphology of scaffolds was characterized by scanning electron microscopy. Samples were mounted onto an aluminum stud and sputter-coated with gold before being examined on a scanning electron microscopy (S-3400, Hitachi, Japan). 2.4.2. The determination of the porosity of scaffolds The porosity of scaffolds was measured using a specific gravity bottle based on Archimedes’ Principle. Briefly, a specific gravity bottle was filled with ethanol (density e ) and weighed (W1 ). The scaffold sample (weight Ws ) was put into the above gravity bottle under vacuum to remove any trapped air in the pores. Then the gravity bottle was filled with ethanol and all the overflowed ethanol was cleaned away carefully (weight W2 ). Finally after the ethanolscaffold was taken out, the gravity bottle was weighed again (W3 ). The porosity of scaffold was determined as follows: (W2 − W3 − Ws )/e × 100 (W1 − W3 )/e

2.1. Material

Porosity (%) =

Poly(lactide-co-glycolide) (PLGA, LA:GA = 50:50, Mn = 100 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd. (Shandong, China). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was provided by Rebone Biomaterials Co., Ltd. (Shanghai, China). Toluidine blue zinc chloride double salt, Human BMP-2 ELISA Kit and fluorescein isothiocyanate-Phalloidin (FITCPhalloidin) were purchased from Sigma–Aldrich (St. Louis, USA). Anti-BMP-2 antibody was obtained from R&D systems Inc. (Minneapolis, USA). Goat anti-mouse lgG H&L (Alexa Flour 594) was from Abcam, Inc. (Cambridge, UK). All cell-culture related reagents were available from Gibco (Grand Island, USA).

where (W1 − W3 )/e is the total volume of the scaffold including pores, (W2 − W3 − Ws )/e is the pore volume in the scaffold.

2.2. Preparation of PLGA scaffold Porous PLGA scaffolds were prepared by combination of phase separation and salt leaching method [25]. Briefly, sieved sodium chloride particles of 200–400 ␮m diameter were added to the PLGA solution (10%, w/v) in 1,4-dioxane. The ratio of sodium chloride:PLGA was 5:1 (w/w). The blend was mixed briefly to form homogeneous slurry and was maintained at −80 ◦ C over 24 h to perform solid–liquid phase separation completely in Teflon molds. After the solvent was removed completely under lyophilization, the sodium chloride was leached out in deionized water. The deionized water was renewed every 3 h until no chloric ion could be detected by dropping of AgNO3 aqueous solution. The fabricated PLGA scaffolds were dried and kept in a desiccator for usage. 2.3. Surface immobilization of 26SCS 26SCS was synthesized as previously described [21] and characterized by FTIR (Nicolet 380, Thermo, USA) and 13 C NMR (AVANCE 400, Bruker, Germany) (Fig. S1, Supporting information). 26SCS immobilization was performed after the aminolysis of PLGA scaffolds according to the method of Croll et al. [26]. In brief,

2.4.3. Atomic force microscopy PLGA film was fabricated by a solution casting technique [25]. Briefly, 10% (w/v) PLGA solution was cast into a Teflon mold. The formed film was removed from the mold until the solvent had thoroughly evaporated under vacuum at 37 ◦ C. Then the films were aminolyzed and immobilized with 26SCS using the method mentioned above. The resulting PLGA films were also named as PLGA, S-PLGA0.05 , S-PLGA0.1 and S-PLGA0.2 respectively. The topographical studies on the surface of above mentioned films were carried out using atomic force microscopy (Veeco/DI, USA). 2.4.4. Determination of contact angle A static contact angle measurement system (Datephysics OCA20, Germany) was used to determine the hydrophilicity of the top surface of PLGA, S-PLGA0.05 , S-PLGA0.1 and S-PLGA0.2 films. Approximately 1 ␮L of water was dropped on the top surface of the films and the contact angle measurement was made after astatic time of 30 s. Five independent determinations at different sites of a film were averaged and each sample was assayed in triplicate. 2.5. Ninhydrin assay The ninhydrin analysis method was employed to qualitatively detect amino groups on the surface of aminated PLGA scaffolds (PLGA0.05 , PLGA0.1 , PLGA0.2 ) [27]. Briefly, the scaffold was immersed in 1 mL of 1.0 mol/L ninhydrin–ethanol solution for 1 min and then placed onto a glass dish and incubated at 37 ◦ C for almost 1 h. The purple red color developed on the scaffolds indicated the presence of amino groups onto scaffolds.

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To further determine the levels of amino groups, the ninhydrin colorimetric assay was performed. Briefly, the scaffold was immersed in 1.0 mL of 1.0 mol/L ninhydrin–ethanol solution for 5 min and heated to 75 ◦ C for 15 min. After cooling, 2 mL of tetrahydrofuran was added to dissolve the scaffolds and ninhydrin reaction products. Simultaneously, known amount of ED standards was also incubated in similar conditions. The absorbance of each sample and standard solution was measured at 568 nm using a microplate reader (SPECTRAmax 384, Molecular Devices, USA) and the levels of amino groups on scaffolds can be quantified according to the standard curve. All samples were tested in triplicate and expressed as mean ± standard deviation (mean ± SD). 2.6. Toluidine blue assay The immobilization of 26SCS on scaffold was visualized by toluidine blue (TB) staining [28]. The PLGA, S-PLGA0.05 , S-PLGA0.1 and S-PLGA0.2 scaffolds were incubated for almost 1 h in the TB solution (0.005% TB solution in 0.01 M hydrochloric acid with 0.2% (w/v) sodium chloride) at room temperature. The purple color developed on the scaffolds indicated the presence of immobilized 26SCS. The 26SCS content on the scaffolds was quantified by TB colorimetric assay [17]. The PLGA and S-PLGA scaffolds were placed in 0.5 mL PBS, followed by 0.5 mL of TB solution. After 1 h of vibration, 1 mL of hexane was added and mixed by vortex to allow phase separation. Then absorbance of aqueous layers at 631 nm was determined by a microplate reader. According to a standard curve of absorbance at 631 nm with different concentration of 26SCS prepared by the same method, the 26SCS content on the surface of S-PLGA scaffolds can be quantified. At least three replicates were carried out for each group and the results were expressed as mean ± SD. 2.7. X-ray photoelectron spectroscopy (XPS) Analysis PLGA, PLGA0.1 and S-PLGA0.1 scaffolds were set as representatives in XPS assay. The surface chemical composition of each scaffold was investigated by XPS (PH1500C, Japan) equipped with an Argon Ion Gun. The elements present in the sample surface were identified from a survey spectrum recorded over the energy range 0–1100 eV and a resolution of 1.0 eV. For further analysis, high-resolution spectra were recorded from pertinent photoelectron peaks to identify the chemical state of each element. Atomic concentration of each element was calculated by determining the relevant integral peak intensities. All the binding energies (BEs) were referenced to the C 1s neutral carbon peak at 285 eV, to compensate for the effect of surface charging. 2.8. Determination of bound rhBMP-2 PLGA and S-PLGA scaffolds with 10 mm of diameter and 5 mm of height were placed in a 24-well plate. 1 mL rhBMP-2 solution (50 ␮g/mL of rhBMP-2 in pH 7.2 phosphate buffer saline (PBS)) was added into each well. The PLGA scaffolds were incubated in the rhBMP-2 solution for 2 h at 37 ◦ C on a shaker and the supernatants were collected respectively. Then rhBMP-2 bound scaffolds were washed with PBS for three times. All the washing liquid was also collected and mixed with previous supernatant respectively. The amount of rhBMP-2 in the collected solution was assayed using a Human BMP-2 ELISA Kit according to manufacturer instructions. Binding efficiency (BE) of rhBMP-2 to the PLGA scaffolds was evaluated according to the following formula: BE (%) =

W − W  a b Wa

× 100

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where Wa and Wb were the mass of rhBMP-2 in PBS solution before and after the incubation of PLGA scaffolds. Each sample was assayed in triplicate (n = 3). 2.9. In vitro release of rhBMP-2 Briefly, 25 ␮g of rhBMP-2 was directly dropped onto the PLGA and S-PLGA scaffolds (S-PLGA0.05 , S-PLGA0.1 and S-PLGA0.2 ) under vacuum supply respectively. Then various rhBMP-2 loaded scaffolds as above mentioned were respectively immersed in 2 mL PBS (pH = 7.4) at 37 ◦ C on a shaker. At a series of predetermined time points, 300 ␮L of the buffer was collected and stored at −80 ◦ C. The collected release buffer was replaced with an equal volume of fresh PBS. For measurement of rhBMP-2 concentration, a Human BMP-2 ELISA Kit was used, following the manufacturer’s instructions. The cumulative release of rhBMP-2 from scaffolds was calculated by the following equation: Cumulative release of rhBMP − 2 (%) =

m  n

m0

× 100

where mn is the mass of rhBMP-2 released from the scaffolds at a certain time and m0 is the mass of total loaded protein. All samples were tested in triplicate (n = 3) and expressed as mean ± SD. 2.10. Cell culture The mouse myoblast cell line C2C12, with osteoblastic potential, was purchased from the American Type Culture Collection (ATCC). C2C12 cells were cultured in 37.5 cm2 flasks with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 ␮g/mL streptomycin at 37 ◦ C, 5% CO2 , in a humidified environment. 2.10.1. Cell attachment C2C12 cells were seeded on the surface of the PLGA, S-PLGA0.05 , S-PLGA0.1 , S-PLGA0.2 scaffolds in a 24 well culture plate at a density of 1.0 × 104 cells/mL. After 24 h incubation, samples (n = 3) were washed with PBS twice and fixed with 1% glutaraldehyde. The fixed cells were washed with PBS twice, and then stained with FITC-Phalloidin for 40 min. DAPI (4 ,6-diamidino-2-phenylindole) solution was added to stain cell nuclei for 10 min. 3D images of the scaffold surface layer were observed using confocal laser scanning microscope (CLSM, Nikon A1R, Japan) and analyzed by the Nikon NIS Elements and Viewer Software. 2.10.2. Cell viability The extracts of various scaffolds were prepared in advance under sterile environment. Briefly, the PLGA and S-PLGA scaffolds with 10 mm of diameter and 5 mm of height were respectively immersed in 24-well plate with serum-free DMEM at 37 ◦ C for 1 d and 4 d respectively. At predetermined time points, the extracts were collected and stocked at −80 ◦ C for further experiments. All samples were tested in triplicate. The obtained extracts were used in MTS assay to evaluate the cell viability. After C2C12 cells at a density of 5000 cells/well were evenly seeded and attached onto a 96-well culture plate, 200 ␮L extracts of scaffolds was added to each well and incubated for 48 h. Standard culture media was used as a positive control. Then the relative cell viability was determined by MTS. Briefly, the medium was replaced with fresh medium containing the tetrazolium dye MTS according to the manufacturer’s procedure. After 1.5 h incubation at 37 ◦ C, solution absorbance was measured at 490 nm by a microplate reader (SPECTR Amax 384, Molecular Devices, USA). Cell viability was calculated by normalizing the absorbance of samples at 490 nm to that of the control. Each sample was assayed in triplicate and mean ± SD values were reported.

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Table 1 The porosity of scaffolds and its surface roughness and water contact angle. Scaffolds

PLGA

Porosity RMS (nm) Ra (nm) WCA (◦ )

85.2 5.50 3.90 86.9

S-PLGA0.05 ± ± ± ±

1.0 0.15 0.16 2.8

86.0 6.73 4.05 79.4

± ± ± ±

1.5 0.43 0.34 1.7

S-PLGA0.1 84.8 10.35 7.67 70.6

± ± ± ±

1.3 0.87 0.67 3.9

Table 2 Atomic compositions of PLGA, PLGA0.1 and S-PLGA0.1 determined via XPS.

S-PLGA0.2 86.3 28.66 23.92 60.4

± ± ± ±

1.6 0.55 0.51 1.1

2.10.3. Determination of alkaline phosphatase (ALP) activity After loading the same amount of rhBMP-2 on scaffolds and lyophilizing, the extracts of PLGA/rhBMP-2 and S-PLGA/rhBMP-2 scaffolds at day 4 were prepared according to the method in Section 2.10.2. PLGA and S-PLGA scaffolds without binding rhBMP-2 were set as negative controls. The bioactivity of rhBMP-2 released from the PLGA and S-PLGA scaffolds was measured by determining its ability to induce ALP activity in C2C12 cells after 5 days culture with the extracts of scaffolds. In brief, C2C12 cells were seeded in 96-well culture plate (1 × 104 cells/well) and allowed to attach overnight. The culture medium was replenished the next day with the extracts collected at day 4 respectively. ALP activity was measured according to standard procedures [29] and normalized to the total protein content. All the experiments were performed in triplicate. 2.10.4. Immunofluorescence assay of rhBMP-2 localized on the surface of C2C12 cells PLGA and S-PLGA scaffolds loaded with same amount of rhBMP2 were prepared to observe the interactions of rhBMP-2 and its receptors on cell surface. After cultured 12 h under the treatment of various scaffolds, C2C12 cells were fixed with 1% glutaraldehyde for 10 min. 1% BSA solution was added as the blocking buffer for 1 h at 37 ◦ C. After washing with PBS twice, cells were incubated with anti-BMP-2 antibody for 1 h at 37 ◦ C and the cultures were placed at 4 ◦ C for 1 h. For rhBMP-2 staining, cells were incubated with a dilution of fluorescently conjugated goat-anti-mouse IgG for 1 h at room temperature. DAPI solution was added to stain cell nucleus for 10 min. rhBMP-2 and cell nucleus in all groups were monitored with confocal laser scanning microscope under the same parameter settings. 2.11. Statistical analysis All numerical date were expressed as the mean ± standard deviation. Statistical analysis was performed with one-way analysis of variance (ANOVA). A value of p < 0.05 was considered as statistical significance. 3. Results 3.1. Morphology characterization As indicated by the SEM micrographs in Fig. 1A–H, the surface morphology structure of PLGA and S-PLGA scafffolds had no obvious difference. All of the scaffolds possessed about 85% of porosity (Table 1), as well as mainly 200–400 ␮m pore size which depended on the granule size of porogen NaCl. Fig. 1I–L shows the AFM images (5.0 ␮m × 5.0 ␮m) of PLGA, SPLGA0.05 , S-PLGA0.1 and S-PLGA0.2 films. The virgin PLGA films had a relatively smooth and uniform surface. However, the films became progressively rougher for S-PLGA0.05 , S-PLGA0.1 and S-PLGA0.2 films. The root mean square roughness (RMS) value and arithmetic average roughness (Ra ) were used to quantitatively characterize the roughness of surface [30,31]. For these samples, the RMS value was calculated to be 5.50 ± 0.15 nm for PLGA films, while increased to 6.73 ± 0.43 nm, 10.35 ± 0.8 nm and 28.66 ± 0.55 nm for S-PLGA0.05 ,

Sample

C (at.%)

O (at.%)

N (at.%)

S (at.%)

C/O (%)

PLGA PLGA0.1 S-PLGA0.1

30.09 31.15 31.20

69.91 67.72 66.91

0 1.05 1.44

0 0.08 0.44

0.43 0.46 0.47

S-PLGA0.1 and S-PLGA0.2 films, respectively. The same increasing trend of Ra could be seen in Table 1. Hydrophilicity of the PLGA, S-PLGA0.05 , S-PLGA0.1 and S-PLGA0.2 films was identified by water contact angle measurement system, as shown in Table 1. The contact angle of S-PLGA0.2 against water (60.4 ± 1.1◦ ) was relatively smaller than that of S-PLGA0.1 , S-PLGA0.05 and PLGA (70.6 ± 3.9◦ , 79.4 ± 1.7◦ , 86.9 ± 2.8◦ , respectively). It is known that PLGA is hydrophobic [8] and the synthesized 26SCS are hydrophilic attributed to the rich hydroxyl groups and sulfonic acid groups in the molecule. As a result, surface hydrophilicity of S-PLGA scaffold was improved by 26SCS immobilization. It is also reported that scaffold with a hydrophilic surface is more favorable for cell attachment and proliferation [25] as well as the binding of some proteins [32]. 3.2. XPS analysis Changes in the surface chemistry of different PLGA scaffolds can be monitored by determining their relative elemental compositions by XPS. PLGA, PLGA0.1 and S-PLGA0.1 scaffolds were set as representatives in XPS analysis. The survey spectra of the PLGA, PLGA0.1 and S-PLGA0.1 scaffolds are displayed in Fig. 2A, showing the carbon (C 1s) and oxygen (O 1s) photoelectron signals [33]. As seen, there were few changes in the survey spectra of different scaffolds. The atomic compositions calculated from the XPS spectra for PLGA, PLGA0.1 and S-PLGA0.1 scaffolds surface are detailed in Table 2, showing a slight increase in the carbon/oxygen (C/O) ratio for the PLGA0.1 and S-PLGA0.1 samples. What is more, within the XPS sampling depth, PLGA0.1 scaffolds contained approximately 1.05 at.% nitrogen, while S-PLGA0.1 scaffolds contained 1.44 at.% nitrogen and 0.44 at.% sulfur. As the existence of N atoms in 26SCS molecules, the aminolyzed scaffolds with 26SCS treatment had a little bit higher N atomic composition than the scaffolds just with ED treatment. Since the chemical states of the carbon, nitrogen and sulfur atoms in the scaffolds could be key indicators of the chemical changes that occur upon the treatments in question, high resolution C 1s, N 1s, S 2p spectra were applied to further characterize the variation of surface elemental compositions of scaffolds. The high resolution C 1s spectra of PLGA, PLGA0.1 and SPLGA0.1 scaffolds (Fig. 2B) consisted of three components which were centered at ∼289 eV, ∼287 eV and ∼285 eV and attributed to ester/carboxylic bonds (O C O), ether bonds ( C O ) and carbon-hydrogen bonds ( C H ), respectively [26,32]. Only a slight variation could be seen in chemical bonds groups, indicating aminolysis induced small changes on scaffolds surface (Table S1, supporting information). The high resolution N 1s spectra of PLGA0.1 and S-PLGA0.1 were centered at around 400 eV (Fig. 2C). Both N 1s spectra showed a shoulder at 402 eV, indicating the presence of protonated amine groups (NH+ ) which would be available for further functionalization [33]. The presence of amide or urethane groups ( HN C O) can be mainly attributed to the reaction of the PLGA with ED. The high resolution S 2p photoelectron spectra of the PLGA0.1 and S-PLGA0.1 scaffolds surfaces were centered at 168 eV (Fig. 2D). It is clear that the sulfur concentration of the S-PLGA0.1 scaffolds was much higher than PLGA0.1 scaffolds, suggesting successful immobilization of 26SCS on PLGA scaffolds.

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Fig. 1. Surface characterization of PLGA and S-PLGA scaffolds and films. (A–H) SEM images under 50× and 200×; (I–L) AFM images.

3.3. Determination of amino group and 26SCS on scaffolds Ninhydrin and TB staining methods were developed to qualitatively analyze the distribution of amino group and 26SCS on PLGA and S-PLGA scaffolds (Fig. 2E). With the increasing ED concentration, the color of S-PLGA scaffolds surface deepen to purple red in the Ninhydrin assay, indicating the growing amount of amino groups on the scaffolds. The same phenomena can be seen in toluidine blue assay, the color of S-PLGA0.2 scaffolds showed the deepest purple, suggesting the maximum level of 26SCS immobilized. To further quantify the amount of amino group and 26SCS immobilized on the S-PLGA scaffolds, ninhydrin and TB colorimetric assays were performed (Fig. 2F and G). With the increasing ED concentration, more amount of amino group and 26SCS were immobilized on S-PLGA scaffolds. For S-PLGA0.2 scaffolds, the amount of amino groups immobilized was 1.3 ± 0.1 mM/cm2 , while the immobilized 26SCS was calculated to be 2.2 ± 0.1 ␮g/cm2 .

3.4. Determination of bound rhBMP-2 The binding efficiency of rhBMP-2 to S-PLGA scaffolds was determined and compared with that of PLGA scaffolds, as shown in Fig. 3A. It could be seen that the rhBMP-2 binding efficiency enhanced gradually with the increased ED concentration. The rhBMP-2 binding efficiency of S-PLGA0.2 scaffolds (33.87%) was almost 10 times higher than that of PLGA scaffolds (3.85%). It

demonstrated that introducing 26SCS on PLGA scaffolds surface could effectively enhance rhBMP-2 binding amount of the scaffold. 3.5. In vitro release of rhBMP-2 The release profiles of rhBMP-2 from PLGA and S-PLGA scaffolds were identified by Human BMP-2 ELISA Kit, as shown in Fig. 3B. It could be seen that each scaffold showed a burst release of rhBMP-2 at the first day, followed by a relatively slow release. However, for S-PLGA scaffolds, a lower initial release pattern was found. Especially for S-PLGA0.2 scaffolds, there was almost 30% lower release level compared to the PLGA scaffold at the first day. This was mainly due to the presence of surface capturers (26SCS) which had strong interaction with rhBMP-2. Specifically, with the increasing amount of immobilized 26SCS by ED, enhanced electrostatic interaction between rhBMP-2 and scaffolds was formed and reduced initial release amount of rhBMP-2 was further achieved for S-PLGA scaffolds. While at the later period of release process, with surface capturers gradually falling off, S-PLGA scaffolds tended to be more and more similar to PLGA scaffolds. Thus when incubating time extended, S-PLGA scaffolds can achieve closer cumulative release amount of rhBMP-2 but with a lower initial burst release compared to PLGA scaffolds. 3.6. Cell attachment and viability Cell attachment is one of the most important behaviors that control cell–biomaterial interactions. Fig. 4A displays

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Fig. 2. Chemical composition analysis of PLGA and S-PLGA scaffolds surface. (A–D) Representative XPS spectra of PLGA, PLGA0.1 and S-PLGA0.1 scaffolds surface; (E–G) determination of amino group and 26SCS on scaffolds surface by ninhydrin and toluidine blue staining assay, *p < 0.01. (0, 0.05, 0.1, 0.2 represented for the ED concentration).

representative microscopy images of cells grown on PLGA, SPLGA0.05 , S-PLGA0.1 and S-PLGA0.2 scaffolds after cultured for 24 h. It could be seen that the PLGA scaffold was highly hydrophobic and supported low levels of cell attachment. While with the increased ED concentration, an increasing number of cells were attached onto S-PLGA scaffolds around the pores. What is more, C2C12 cells on the S-PLGA scaffolds retained their elongated, spindle-shaped

morphology especially S-PLGA0.2 scaffolds, as shown in the magnified images. Whereas, C2C12 cells were weakly spread with a round shape actin cytoskeleton on the PLGA scaffolds without modification. As shown in Fig. 4B, MTS assay demonstrated that the overall viability of cells remained high (>100%), indicating all the scaffolds were of excellent biocompatibility.

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Fig. 3. (A) Binding efficiency of rhBMP-2 to PLGA and S-PLGA scaffolds, *p < 0.05, compared with PLGA scaffold; (B) cumulative release of rhBMP-2 from PLGA/rhBMP2 and S-PLGA/rhBMP-2 scaffolds.

3.7. Bioactivity assay of released rhBMP-2 Cells incubated in the extracts of PLGA/rhBMP-2 and SPLGA/rhBMP-2 scaffolds at day 4 showed an effective up-regulation in ALP activity after 5 days of C2C12 culture compared with the negative control (Fig. 5A). PLGA and S-PLGA scaffolds before binding rhBMP-2 showed a similar ALP activity. After binding rhBMP-2, the medium containing released rhBMP-2 from S-PLGA0.1 and SPLGA0.2 scaffolds induced a significant enhancement in ALP activity, while S-PLGA0.05 scaffolds demonstrated a similar ALP activity with PLGA. 3.8. Effect of immobilized 26SCS on the rhBMP-2 binding to cell surface In order to further investigate the effect of immobilized 26SCS on rhBMP-2 activity, we examined the binding efficiency between rhBMP-2 and its receptor under the treatment of S-PLGA scaffolds. Hence, the detectable amount of rhBMP-2 localized on cell layer reflected rhBMP-2 signal intensity directly. RhBMP-2 was detected by immunofluorescence staining via anti-BMP-2 primary antibody and fluorescently labeled goat-anti-mouse IgG. Cell nucleus was stained with DAPI served for cell localization. Fig. 5B shows that rhBMP-2 signaling was intensively enhanced and spindled with the increasing amount of 26SCS immobilized onto scaffolds. The result suggested that the immobilized 26SCS would increase the binding efficiency between rhBMP-2 and its receptor. 4. Discussion In our study, 26SCS immobilized PLGA scaffolds were developed to further potentiate the bone repair performance. The

Fig. 4. Cell morphology and cell viability of C2C12 on the scaffolds. (A) Observation of cytoskeleton stained with FITC-Phalloidin (green) and nuclei stained with DAPI (blue) of cells under 200× and 600×; (B) cell relative viability of C2C12 determined by MTS assay. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

immobilization of 26SCS onto the scaffold surface applied electrostatic interaction between negatively charged sulfonic groups ( SO3 − ) of 26SCS and the positively charged amino residues ( NH2 ) on aminolyzed scaffolds surface. This process also allowed the sulfonic groups of 26SCS to subsequently trap positively charged rhBMP-2 [34] when binging rhBMP-2 on scaffolds. After 26SCS immobilizing, surface topography became rougher with the increased ED concentration, while the interconnected pore structure remained the same in all scaffolds (Fig. 1). Moreover, there is a little difference in the chemical composition between modified and control scaffolds (Table 2). These revealed the reaction condition was mild and made a little change to scaffolds. The S-PLGA scaffolds developed in this study had several advantages for bone regeneration (Fig. 6). One of the key contributions in

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Fig. 5. (A) ALP activity of PLGA and S-PLGA scaffolds before and after binding rhBMP-2, *p < 0.05, compared to PLGA scaffolds after binging rhBMP-2; (B) effect of S-PLGA scaffolds on the binding efficiency between rhBMP-2 and its receptor, rhBMP-2 was detected with anti-BMP-2 antibody and fluorescently labeled goat anti-mouse IgG (orange-red) and cell nuclei were stained with DAPI (blue) for cell orientation (600×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

the present work was the improvement of cellular affinity owing to the favorable superficial properties. Khan et al. demonstrated that polysaccharide-based materials had good biocompatibility with acceptable host response, and had the ability to promote cell adhesion and proliferation [13]. In our study, S-PLGA scaffolds, with the anchoring of 26SCS (a heparin-like polysaccharide) on the surfaces, were proved to be more favorable for cell attachment and growth (Fig. 4). The improved hydrophilicity and roughness of the S-PLGA scaffolds might have contributed to these results for its increased cell recognition sites [35]. The other crucial merit taking into account was that S-PLGA scaffolds could achieve high binding efficiency and controlled release of rhBMP-2. In our study, S-PLGA scaffolds can bind rhBMP-2 more rapidly and easily (Fig. 3A), which was one of the most important capacities for bone repairing materials. While in vitro release of rhBMP-2, S-PLGA scaffolds exhibited a lower burst release compared with PLGA scaffolds and afterwards

ensured a sustained release for 15 days (Fig. 3B). Many previous investigations reported that some amount of early release combined with sustained release is favorable for bone regeneration [36,37]. In our study, the release pattern of rhBMP-2 from S-PLGA scaffolds was very similar as reported [38] and should be better for promoting bone regeneration. We rationalized that intermolecular electrostatic interaction was the dominant force binding rhBMP-2 onto S-PLGA scaffolds. Moreover, BMP-2 might recognize specific sulfation motifs in 26SCS chains and interact with 26SCS, thus leading to a sustained release of rhBMP-2. Another significant benefit to develop S-PLGA scaffolds was its ability to achieve higher activity of the released rhBMP-2. Although lots of efforts have been made to develop BMP-2 controlled delivery systems in bone tissue engineering, many of these continued to have limitations to achieve active release of BMP-2 [39]. Based on the results of in vitro ALP activity, rhBMP-2 released from S-PLGA scaffolds revealed a higher activity than PLGA scaffolds

Fig. 6. Schematic diagram of the S-PLGA/rhBMP-2 scaffolds on bone regeneration. (1) The 26SCS assembled onto the scaffolds surface enhanced the cell attachment. (2) The electrostatic interaction between rhBMP-2 and 26SCS onto the S-PLGA scaffolds lead to the enhanced the absorption and controlled release of rhBMP-2. (3) The bioactivity of rhBMP-2 was enhanced by its synergistic action with released 26SCS. The released 26SCS enhanced binding efficiency between rhBMP-2 and its receptors. Consequently, the enhanced cell attachment, controlled release and rhBMP-2 bioactivity promoted the bone regeneration.

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(Fig. 5A). Surprisingly, the ALP activity did not entirely match with the releasing amount of rhBMP-2. When loaded the same amount of rhBMP-2, PLGA scaffolds were expected to release a higher amount of rhBMP-2 at day 4 than S-PLGA scaffolds according to the cumulative release profile (Fig. 3B). Nevertheless, ALP activity of PLGA scaffolds was lower than S-PLGA scaffolds in spite of its higher releasing amount of rhBMP-2. The reason for this phenomenon may be that S-PLGA scaffolds could increase the binding efficiency between rhBMP-2 and its receptor (Fig. 5B) and enhance rhBMP-2 bioactivity. This enhancement associated with the S-PLGA scaffolds exactly coincided with our previous study. Our previous study [21,22] demonstrated that 26SCS was a potent enhancer of rhBMP-2 bioactivity and a synergistic factor of rhBMP-2 for bone regeneration. What was more surprising was that the protein Noggin as a BMP-2 antagonist failed to block the function of BMP-2 in the presence of 26SCS according to the research [21]. The findings supported the concept that immobilized 26SCS can enhance rhBMP-2 bioactivity. That is to say, in the rhBMP-2 release process of S-PLGA scaffolds, the electrostatic interaction weakened and emitted the immobilized 26SCS. Then the dissociative 26SCS played the synergic action with the released rhBMP-2 to enhance its bioactivity. 5. Conclusion In this study, PLGA scaffolds surface was immobilized with 26SCS via electrostatic assembly. The immobilized 26SCS was found to improve surface hydrophilicity and cell attachment. In addition, the sustained release pattern of rhBMP-2 was achieved due to the affinity between 26SCS and rhBMP-2. Furthermore, the synergistic effect between rhBMP-2 and the immobilized 26SCS lead to a higher bioactivity of released rhBMP-2. These observations indicated 26SCS immobilized scaffolds could be a promising carrier for osteogenesis. Acknowledgments The authors are indebted to the financial support from the National Basic Research Program of China (973 Program, 2012CB933600), the National Natural Science Foundation of China (Nos. 31273011 and 31330028), the National Science and Technology Support Program (2012BAI17B02), and the Program for New Century Excellent Talents in University (NCET-12-0856). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.07.012.

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