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mesoporous bioactive glass scaffold via polydopamine
Accepted Manuscript Title: Interfacial reinforcement in a poly-l-lactic acid/mesoporous bioactive glass scaffold via polydopamine Authors: Yong Xu, Ping Wu, Pei Feng, Wang Guo, Wenjing Yang, Cijun Shuai PII: DOI: Reference:
S0927-7765(18)30365-5 https://doi.org/10.1016/j.colsurfb.2018.05.065 COLSUB 9384
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
6-2-2018 28-5-2018 29-5-2018
Please cite this article as: Yong Xu, Ping Wu, Pei Feng, Wang Guo, Wenjing Yang, Cijun Shuai, Interfacial reinforcement in a poly-l-lactic acid/mesoporous bioactive glass scaffold via polydopamine, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.05.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interfacial reinforcement in a poly-l-lactic acid/mesoporous bioactive glass scaffold via polydopamine Yong Xu 1,2,#, Ping Wu 3,#, Pei Feng 1, Wang Guo 1, Wenjing Yang 1, Cijun Shuai 1,4,5,* 1 State Key Laboratory of High Performance Complex Manufacturing, College of Mechanical and Electrical Engineering, Central South University, Changsha 410083, China
China 3 College of Chemistry, Xiangtan University, Xiangtan 411105, China
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4 Jiangxi University of Science and Technology, Ganzhou 341000, China
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2 Department of Mechanical and Energy Engineering, Shaoyang University, Shaoyang 422004,
5 Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha 410008, China
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# These authors contributed equally to this work
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* Correspondence: [email protected]; Tel: +86-731-84805412; Fax: +86-731-88879044
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Graphical abstract
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Caption: Graphical abstract demonstrated the preparation and performance tests of poly-l-lactic acid (PLLA)-based scaffolds. Polydopamine modified mesoporous bioactive glass (p-MBG) was prepared by oxidation self-polymerization of dopamine in a tris(hydroxymethyl) aminomethane hydrochloride buffer solution (Tris-HCL). Scaffolds were prepared by selective laser sintering process (SLS).
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Highlights
PLLA/p-MBG scaffold was constructed by selective laser sintering.
Polydopamine was used as a cross-linking bridge to improve interfacial interaction.
PLLA/p-MBG scaffold has improved mechanical properties and hydrophilicity.
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PLLA/p-MBG scaffold also has good biocompatibility.
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Abstract
The weak interfacial interaction between polymer and bioceramic leads to a low reinforcing
effect, even though polymer/bioceramic scaffolds are considered potential implants. In this work, a porous poly-l-lactic acid (PLLA) scaffold containing polydopamine-modified mesoporous bioactive glass (p-MBG) was fabricated by selective laser sintering. The polydopamine was introduced as a cross-linking bridge between MBG and PLLA to improve the interfacial
interaction between of them. On the one hand, the amine groups of polydopamine formed strong hydrogen bonds with the hydroxyl groups of MBG. On the other hand, the catechol groups of polydopamine formed strong hydrogen bonds with the ester groups of PLLA. The PLLA/p-MBG scaffold showed higher compressive strength and modulus (62.9 MPa and 3.6 GPa, respectively) than the PLLA/MBG scaffold (41.9 MPa and 2.1 GPa, respectively). This result was explained by the fact that the enhanced interfacial adhesion promoted the dispersion of p-MBG and increased
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the efficiency of stress transfer in the matrix. Additionally, the PLLA/p-MBG scaffold showed good cytocompatibility. This study suggested that the PLLA/p-MBG scaffold may have a potential
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application in tissue engineering.
Keywords: cross-linking bridge; interfacial reinforcement; cytocompatibility; mesoporous bioactive glass; polydopamine
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1 Introduction
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Incorporating bioceramic into a polymer scaffold to improve the biological and mechanical
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properties has attracted a considerable amount of attention. Nevertheless, the mechanical properties of the scaffold were decreased at high filler loading due to the weak interfacial
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interaction between the bioceramic and polymer resulting from their significant difference in
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physicochemical properties [1-3]. The weak interfacial interactions result in a morphological defect or failure at the interface, causing deterioration of the mechanical properties of the scaffold [4, 5]. Therefore, it is of great importance to enhance the interfacial interaction between the
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polymer and bioceramic. Several researchers have been committed to the enhancement of the interfacial interaction between bioceramics and polymers [6-8]. As reported by Rezabeigi E et al.,
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45S5 bioactive glass modified by 3-methacryloxypropyltriethoxysilane exhibited good dispersion in polylactic acid, and the polylactic acid/bioglass scaffold had improved mechanical properties [9]. Fang Z et al. reported that using γ-aminopropyltriethoxysilane to modify hydroxyapatite (HA)
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could improve the dispersion and interfacial compatibility of HA in poly-l-lactic acid (PLLA), which improved the mechanical properties of the HA/PLLA scaffold [10]. Nevertheless, these methods might have potential side effects due to the use of toxic chemicals. Therefore, it is necessary to develop a safe and effective modification strategy for improving the interfacial interaction between the bioceramics and polymers. Dopamine (C8H11O2N, 4-(2-aminoethyl) benzene-1,2-diol) is a type of cell signal agent widely
found in organisms. It can self-polymerize to form polydopamine, which has a similar structure to the protein melanin [11]. Recently, the application of polydopamine in tissue engineering has drawn keen attention due to its excellent biocompatibility and biodegradability [12, 13]. More importantly, polydopamine can form non-covalent crosslinks with matrices that contain ester and hydroxyl groups [14]. PLLA is a type of polyester polymer with good biocompatibility. The ester groups in its molecular chain may form strong hydrogen bonds with the catechol groups of
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polydopamine. Mesoporous bioactive glass (MBG) is a newly developed bioactive material. The hydroxyl groups enriched on its mesoporous channels also may form strong hydrogen bonds with
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the amine groups of polydopamine [15]. Therefore, polydopamine is expected to establish a cross-linking bridge between PLLA and MBG, allowing the mechanical properties of the scaffold to be improved while retaining its good biological properties.
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In this work, to address the weak interfacial interactions between mesoporous bioactive glass
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(MBG) and poly-l-lactic acid (PLLA), polydopamine was introduced as a cross-linking bridge
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between of them. The polydopamine-modified mesoporous bioactive glass (p-MBG) was prepared based on the self-polymerization of dopamine and the mesoporous structure was analyzed.
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Moreover, a porous PLLA scaffold containing p-MBG was fabricated by selective laser sintering.
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The compressive strength and modulus of scaffold were measured. The micromorphology of p-MBG and its dispersion in scaffold were investigated. In addition, the mechanisms of interfacial interaction between p-MBG and PLLA were analyzed. Besides, the cytocompatibility and
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hydrophilicity of scaffold were also evaluated.
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2 Materials and methods
2.1 Materials and preparation Poly-l-lactic acid (PLLA, Mn 150,000; inherent viscosity 2.5~3.0 dl/g in chloroform at 25℃)
was kindly provided by Shenzhen Polymtek Biomaterial Co., Ltd. (Shenzhen, China). Dopamine
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hydrochloride and tris(hydroxymethyl) aminomethane (Tris, 99%) were purchased from Sigma and used as received. MBG was prepared by a sol-gel method process, according to the research reported by Chang J et al. [16]. The modification of MBG was performed by the oxidative polymerization of dopamine under alkaline conditions. Briefly, 4 g of MBG was dispersed in 1000 mL Tris-HCl buffer solution (10 mM, pH 8.5) and treated ultrasonically for 30 min. Subsequently, 2g of dopamine hydrochloride was added to the solution and mildly stirred for 5h at ambient
temperature. Then, the solution was filtrated and rinsed with deionized water 3 times to remove the polydopamine that was not adhered on the MBG. Finally, a dark brown p-MBG powder was obtained after drying overnight at 60 °C. To prepare the blend powder, a predetermined amount of p-MBG or MBG was separately added to beakers containing 50 ml of absolute ethanol and then sonicated for 30 min. Subsequently, an appropriate amount of PLLA was added to the suspension and treated with ultrasonic vibration and magnetic stirring for another 30 min. The suspension was
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then filtered, dried at 60 °C for 12 h and mechanically ground to obtain a mixed powder.
The scaffold was fabricated via selective laser sintering. During the preparation, the laser
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beam selectively scanned the powder on the sintering platform according to the slice profile of the scaffold. The laser energy made the temperature of the powder rise sharply to the melting point, which caused the powder to melt and rapidly solidify. Subsequently, the
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sintering platform was moved down a powder layer thickness and the sintering process was
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repeated. The process parameters remained constant throughout the sintering: laser power of
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2.3 W, spot diameter of 50 μm, scan rate of 100 mm/s, scan line spacing of 1 mm and powder layer thickness of 0.1 mm. After the sintering was completed, compressed air was used to
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remove the unsintered powder and clean the scaffold.
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2.2 Characterization
The micromorphology of p-MBG was characterized by scanning electron microscopy (SEM, Tescan Mira3 Lmu, Co., Czechia). N2 adsorption–desorption isotherms were obtained on a
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Beshide 3H-2000PS2 at 200 °C under continuous adsorption conditions. The surface area and pore size distribution of p-MBG powder were analyzed by the Brunauer-Emmett-Teller (BET) and
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Barrett-Joyner-Halenda (BJH) method. The chemical composition of p-MBG was characterized by Fourier transform infrared spectrophotometer (FTIR, Thermo Scientific Co., Madison, WI, USA) and X-ray photoelectron spectroscopy (XPS, ThermoFisher-VG Scientific, USA). For
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thermo-gravimetric (TG) analysis, 8-10 mg of sample powder was placed in ceramic crucible and heated from environment temperature to 800 °C within a TG analyzer (Nanjin Dazhan Institute of Electromechanical Technology STA-200, China), at a heating rate of 20 °C/min in nitrogen condition. The micromorphology of the scaffold was characterized via SEM to analyze the distribution of p-MBG. The compressive strength and modulus of the scaffold were tested using a universal
mechanical testing machine (Shanghai Zhuoji Instruments Co., Ltd., Shanghai, China) at a cross-head speed of 0.5 mm/min. Each test consisted of five samples for replicates, from which the average value and standard deviation were calculated. The chemical groups of the PLLA/p-MBG scaffold were observed by FTIR to examine the interaction between p-MBG and PLLA. The hydrophilicity of these scaffolds was evaluated via water contact angle measurement with an Attension Theta system (Biolin Scientific Co. Ltd., Stockholm, Sweden). For each sample,
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five measurements were performed, from which the average value and standard deviation were calculated.
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2.3 Cell culture
MG-63 cells (Cellular Biology Institute, Shanghai, China) were selected in cytocompatibility analysis. Disk scaffold samples (10 mm in diameter and 2 mm in height) were used to evaluate the
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effect of the scaffold material composition on cell behavior (adhesion and viability). Cells were
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cultured in DMEM (KeyGEN BioTECH) supplemented with 10% fetal bovine serum (FBS) and 1%
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penicillin/streptomycin sulfate at a density of 1 × 105 cells/scaffold under a humidified atmosphere of 95% air. The culture medium was renewed every two days. All the instruments and scaffold
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used in this experiment were previously UV sterilized for one hour.
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After 3 days of culturing, the cell/scaffold complexes were collected from the culture medium and rinsed with phosphate buffered solution (PBS) for three times. Subsequently, the cell/scaffold complexes were fixed with 3% glutaraldehyde for 30 min and dehydrated with gradient ethanol.
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Finally, the cell/scaffold complexes were dried in air and subjected to gold sputtering to observe the cell morphology under SEM. The viability of the cells was analyzed by staining the viable
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cells and dead cells with calcein AM and propidium iodide (PI), respectively. Briefly, the cell/scaffold complexes were collected from the culture medium and rinsed twice with PBS. Then, cells were removed from the cell/scaffold complexes with trypsin solution and reseeded onto glass
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slides. After 12 h, cells were then incubated in a medium containing 2 μM calcein AM and 4 μM PI for 30 minutes at 37℃. Afterward, optical analysis was performed by a fluorescence microscope fitted with a digital camera. After osteoblast induction culture for 3 days, LabAssay (TM) ALP kit (Wako, Osaka, Japan) was used to detect the level of alkaline phosphatase (ALP) to evaluate osteogenic differentiation of MG-63 cells. Specifically, a 0.25% trypsin solution was used to remove adherent cells. After
then, cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS, followed by ALP staining performed according to the manufacturer's instructions. Finally, images of stained cells were taken by an inverted microscope (TE2000U, Nikon, Japan). 2.4 Statistical analysis The data of experiments were expressed as the mean ± standard deviation and analyzed using Levene’s test. Statistically significant differences between groups were measured using the
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Student-Newman-Keuls post-hoc test (p). The difference was regarded as statistically significant when * p <0.05.
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3 Results and discussions 3.1 Polydopamine modified MBG
The micromorphology and structure schematic of p-MBG are shown in Fig. 1. The color of
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MBG powder changed from white to dark brown after modification, as shown in the inset of Fig.
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1a and b, indicating that polydopamine successfully formed on MBG. In this process, the amine
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groups of dopamine formed hydrogen bonds with the hydroxyl groups of MBG, and then underwent oxidative self-polymerization to form polydopamine on the surface of MBG [17]. As
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observed from the micromorphology in Fig. 1b, some embossments that existed on p-MBG
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surface are potentially due to the coverage of polydopamine. A schematic diagram of hydrogen bonding between polydopamine and p-MBG is shown in Fig. 1c. FTIR was used to analyze the surface chemical composition of powder, as presented in Fig. 1d.
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Absorption peaks appeared at approximately 3500 and 1637 cm-1, which were attributed to the asymmetric stretching vibration band and bending vibration mode of the O-H of absorbed water,
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respectively [18]. The peaks appearing at 1100 and 802 cm-1 were corresponded to the asymmetric stretching vibration band of Si-O-Si and symmetrical stretching vibration band of Si-O, respectively [19]. For p-MBG, the peak at around 3500 cm-1, which may be due to the v(N-H)
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stretching mode of polydopamine, intensified compared to the MBG [20]. In addition, after modification, the peak at 802 cm-1 weakened and a new absorption peak was detected at 1506 cm-1, which may be due to the effects of v(C=N) stretching and N-H bending vibration mode of aromatic rings in polydopamine, respectively [21]. These results further demonstrated that polydopamine successfully formed on the MBG. Wide scan x-ray photoelectron spectroscopy (XPS) of p-MBG particles are shown in Fig. 1 e.
After polydopamine modification, the XPS spectra of p-MBG particles displayed a new peak at 400 eV, which corresponds to N1s. The presence of nitrogen was attributed to amine groups of polydopamine on the surface of p-MBG particles. Thermo-gravimetric (TG) analysis was used to estimate the amount of polydopamine attached on the p-MBG (Fig. 1f). The mass loss in the range of 50-200 °C was attributed to the adsorption of moisture from environment. And the mass loss at temperatures over 200 °C was caused by the decomposition of polydopamine. By calculating the
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amount of coke in samples above 650 °C, it was confirmed that the amount of polydopamine attached on p-MBG was about 12 wt. %.
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The N2 sorption isotherm and corresponding mesopore size distribution of p-MBG are illustrated in Fig. 2. The results indicated that polydopamine modification had no obvious influence on the mesopore structure and size distribution. As shown in Fig. 2a, p-MBG exhibited
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type IV isotherms and the capillary condensation increased from 0.4 to 0.6 of P/P0, which was
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representative of mesopores with relatively narrow pore size distribution [22]. The distribution of
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the corresponding BJH mesopore size in Fig. 2b was obtained from the adsorption branch. p-MBG showed slightly narrow pore size distribution, and the peak pore diameter of p-MBG was 5.487
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nm. In addition, the BET surface area of p-MBG was 396 m2g-1 and the BJH pore volume was
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reduced to 0.541 cm3g-1, as illustrated in Table 1. It was attributed to the occupation of polydopamine on the inner wall of mesopores as well as the disturbation of amidogen groups [23,
3.2 Scaffold
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24]. In conclusion, the mesoporous structure of p-MBG was well preserved.
Elliptical cylindrical scaffolds are exhibited in Fig. 3. Their dimensions were 12×9×8 mm
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(major axis × minor axis × height). Moreover, both the sizes of struts and pores were about 400μm, which is believed to favor the growth and vascularization of new bone [25]. The interconnected porous scaffold could carry out the transport of nutrients and provide physical support for the
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growth of new tissues. This result suggested that the selective laser sintering was an effective strategy for the preparation of bone repair scaffold. FTIR spectra of PLLA, PLLA/p-MBG and PLLA/MBG scaffolds are shown in Fig. 4a. The strong peak at 1758 cm-1 corresponded to the C=O stretching vibration of the ester group in PLLA [26]. For PLLA/p-MBG scaffold, this strong absorption peak shifted toward a lower wavenumber at 1753 cm-1, suggesting an intermolecular interaction between p-MBG and PLLA [27]. This shift
was ascribed to the hydrogen bond formed between catechol groups of p-MBG and ester groups of PLLA [28]. Moreover, a simple hydrogen bond cross-linking sketch diagram is displayed in Fig. 4b, in which polydopamine acts as a cross-linking bridge to connect p-MBG and PLLA. On the one hand, amine groups of polydopamine formed strong hydrogen bonds with the hydroxyl groups of MBG. On the other hand, catechol groups of polydopamine also formed strong hydrogen bonds with the ester groups of PLLA. Polydopamine cross-linking bridge enhanced the interface bonding
scaffold.
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3.3 The micromorphology and mechanical properties of scaffold
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between p-MBG and PLLA, thereby potentially improving the mechanical properties of the
The dispersion of p-MBG and MBG in the scaffold is illustrated in Fig. 5. EDS spectra at the p1 and p2 sites are provided as illustrations in Fig. 5c and f. The emergence of peaks of Si and N
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indicated that these white particles are MBG and p-MBG, respectively. When the content of
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addition was 5 wt. %, both p-MBG and MBG showed uniform dispersion in the PLLA matrix, as
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shown in Fig. 5a and d. However, for PLLA/MBG scaffold, an agglomeration phenomenon (as shown with red arrows) began to occur and became conspicuous as the content increased from 10
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to 15 wt. % (Fig. 5b and c). Comparatively, the dispersion of p-MBG in the PLLA matrix was
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much improved at the same loading levels (Fig. 5e and f). This was because hydrogen bonding formed between the catechol group of p-MBG and the ester group of PLLA, as schematically shown in Fig. 4b, which promoted the dispersion of p-MBG in the matrix [29]. The uniform
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dispersion of the filler in the matrix might be helpful to improve the ultimate mechanical and other properties of the scaffold [30, 31].
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The dependence of the compressive strength and modulus of PLLA/p-MBG and PLLA/MBG
scaffold on filler contents is shown in Fig. 5g and h. Clearly, the introduction of p-MBG and MBG significantly improved the compressive strength and modulus of PLLA scaffold. The compressive
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strength and modulus of PLLA scaffold were 20.8 ± 2.5 MPa and 1.8 ± 0.1 GPa, respectively.
For PLLA/MBG scaffold, the compressive strength and modulus first increased and then decreased as the MBG content increased. The maximum compressive strength and modulus of the PLLA/MBG scaffold were 50.2 ± 4.3 MPa and 3.1 ± 0.2 GPa, respectively, at 10 wt. %. It was attributed to the fact that the high strength and modulus of MBG and the particle enhancement effect had a positive effect on increasing the compressive strength and modulus of the scaffold [32,
33]. Additionally, when the content reached 15 wt. %, the decrease in strength and modulus was caused by the aggregation of MBG in the matrix [34]. For PLLA/p-MBG scaffold, the compressive strength and modulus increased with increasing p-MBG contents. In detail, the compressive strength and modulus increased from 45.3 ± 2.2MPa and 2.3 ± 0.2GPa to 62.9 ± 5.2 MPa and 3.6 ± 0.3 GPa, respectively, as the p-MBG content increased from 5 to 15 wt. % (Fig. 5g and h). In comparison, PLLA/p-MBG scaffold had a higher
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compressive strength and modulus than PLLA/MBG scaffold with the same filler contents. This finding could be explained by the following two aspects. On the one hand, the homogeneous
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dispersion of p-MBG in the PLLA matrix prevented agglomeration, resulting in significant
particle reinforcement [35]. On the other hand, polydopamine acted as a cross-linking bridge between PLLA and p-MBG, which promoted the effective stress transfer at the interface [36].
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3.4 The hydrophilicity of scaffold
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The water contact angle of PLLA/p-MBG scaffold is shown in Fig. 6. Obviously, PLLA
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scaffold exhibited hydrophobic properties with a water contact angle of 105.8 ± 6.4°. The water contact angle of PLLA/MBG scaffold decreased from 82.5 ± 5.3° to 51.6 ± 2.5° as the MBG
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content increased from 5 to 15 wt. %, exhibiting hydrophilic properties. This change indicated that
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the addition of MBG significantly improved the hydrophilicity of PLLA scaffold. Comparatively, PLLA/p-MBG scaffold showed a lower water contact angle at the same loading level, indicating that polydopamine modification could further improve hydrophilicity. There were two probable
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factors responsible for the improved hydrophilicity. On the one hand, even distribution of p-MBG led to an increase in the contact surface area, and hence improved the hydrophilicity of the
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scaffold [37]. On the other hand, polydopamine contained a large amount of hydrophilic groups, such as catechol and amine groups, which might be of benefit to further improve the hydrophilicity [38]. The improved hydrophilicity might be part of the potential contributors to the
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stimulation of cellular response [5]. 3.5 Cell culture Adherent cell morphology after culture for 3 days is shown in Fig. 7. It can be seen that the cells spreading area increased with increasing filler content. Cells were spindle-shaped when the addition content was 5 wt. %, as shown in Fig. 7a and d. Cells extended to a wider range of the scaffold sample surface (Fig. 7b and e) at a filler loading of 10 wt. %. When p-MBG increased up
to 15 wt. %, the adherent cells were fully stretched and diffused along the scaffold (Fig. 7f). In general, cells on PLLA/p-MBG scaffold samples exhibited good attachment morphology, which suggested that these scaffold material were suitable for cell adhesion. There were likely two probable factors that enhanced cell adhesion. On the one hand, polydopamine itself served as an anchor between cell and matrix due to the hydrophilicity and strong adhesion [39]. On the other hand, the uniform dispersion of p-MBG in the scaffold could exhibit more contact surface area to
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provide more attachment sites for cell attachment [40].
The viability of MG-63 cells was assessed by immunofluorescence. After 3 days of culture,
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cells were removed from the cell/scaffold complexes with trypsin solution and reseeded onto glass slides to observe cell morphology, as shown in Fig. 8A. The cells showed good diffusion
morphology and the cell density increased with increasing p-MBG content, indicating that the
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PLLA/p-MBG scaffold material had good cytocompatibility. The cytocompatibility of the scaffold
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material was attributed to the fact that the large specific surface area of the mesoporous structure
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provided cells with numerous adhesion sites. In addition, the OH- and NH2+ groups of polydopamine could improve the hydrophilicity and adjust the surface charge of the scaffold,
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which provided opportunities for the adhesion and proliferation of cells [41]. The ALP activity is
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an early feature of osteoblast differentiation and plays a key role in early bone formation. Hence the cell ALP activity after 3 days of culture was also evaluated and is shown in Fig.8B. It can be seen that the ALP activity of cells increased with the increasing filler loading, suggesting that
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PLLA/p-MBG scaffold material had the ability to stimulate cell differentiation. This was mainly due to the release of active elements (silicon and calcium) from the composite scaffold, which can
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stimulate cell gene expression and mineralization and further induce osteoblast differentiation [42].
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4 Conclusions
In this work, three-dimensional porous PLLA/p-MBG scaffolds for tissue engineering implants
were constructed via selective laser sintering process. Polydopamine was used as the interface compatibilizer to improve the interfacial interaction between MBG and PLLA. BET and BJH analysis showed that the mesoporous structure of p-MBG was well preserved. Polydopamine, as a cross-linking bridge between PLLA and MBG, greatly improved the interfacial compatibility and mechanical properties of PLLA/p-MBG scaffold. Cell culture results indicated that cells
preferentially adhered, proliferated and differentiated on PLLA/p-MBG scaffold, indicating that these scaffold material had good biocompatibility. The results suggest that PLLA/p-MBG scaffold may be a potential candidate for tissue engineering implant applications. Acknowledgements This work was supported by the following funds: (1) The Natural Science Foundation of China (51575537, 81572577, 51705540); (2) Hunan Provincial Natural Science Foundation of China
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(2016JJ1027); (3) The Project of Innovation-driven Plan of Central South University
(2016CX023); (4) The Open-End Fund for the Valuable and Precision Instruments of Central
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South University; (5) The fund of the State Key Laboratory of Solidification Processing in NWPU
(SKLSP201605); (6) National Postdoctoral Program for Innovative Talents (BX201700291); (7) The Project of State Key Laboratory of High Performance Complex Manufacturing, Central South
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University; (8) The Project of Hunan Provincial Science and Technology Plan (2017RS3008); (9)
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The Fundamental Research Funds for the Central Universities of Central South University
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(2018ZZTS145). References
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polylactic acid/Bioglass®; scaffolds produced via nonsolvent induced phase separation, J Biomed Mater Res B, (2016). [10] Z. Fang, Q. Feng, Improved mechanical properties of hydroxyapatite whisker-reinforced poly(L-lactic acid) scaffold by surface modification of hydroxyapatite, Mat Sci Eng C-Mater, 35 (2014) 190-194. [11] F. Scognamiglio, A. Travan, G. Turco, M. Borgogna, E. Marsich, M. Pasqua, S. Paoletti, I. Donati, Adhesive coatings based on melanin-like nanoparticles for surgical membranes, Colloid Surface B, 155 (2017) 553-559. [12] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Mussel-Inspired Surface Chemistry for Multifunctional Coatings, Science, 318 (2007) 426-430. [13] S.K.M. Perikamana, Y.M. Shin, K.L. Jin, B.L. Yu, Y. Heo, T. Ahmad, S.Y. Park, J. Shin, K.M. Park, H.S. Jung, Graded functionalization of biomaterial surfaces using mussel-inspired adhesive coating of polydopamine, Colloid Surface B, 159 (2017) 546-556. [14] J.C. Peeler, S. Schedin-Weiss, M. Soula, M.A. Kazmi, T.P. Sakmar, Isopeptide and ester bond ubiquitination both regulate degradation of the human dopamine receptor 4, J Biol Chem, 292 (2017) jbc.M116.758961. [15] X. Li, L. Zhao, Q. Liang, J. Ye, N. Komatsu, Q. Zhang, W. Gao, M. Xu, X. Chen, Cationic Polyarginine Conjugated Mesoporous Bioactive Glass Nanoparticles with Polyglycerol Coating for Efficient DNA Delivery, J Biomed Nanotechnol, 13 (2017) 280-289. [16] W. Xia, J. Chang, Well-ordered mesoporous bioactive glasses (MBG): a promising bioactive drug delivery system, J Control Release, 110 (2006) 522-530. [17] J. Wang, H. Bai, H. Zhang, L. Zhao, H. Chen, Y. Li, Anhydrous proton exchange membrane of sulfonated poly(ether ether ketone) enabled by polydopamine-modified silica nanoparticles, Electrochim Acta, 152 (2015) 443-455. [18] K. Ding, P. Yang, X. Cheng, Surface Treatment of Cement-Based Materials Using SiO2 Nanoparticles Towards Enhanced Water Absorption Property, J Nanosci Nanotechno, (2017). [19] H. Zhu, C. Hu, F. Zhang, X. Feng, J. Li, T. Liu, J. Chen, J. Zhang, Preparation and antibacterial property of silver-containing mesoporous 58S bioactive glass, Mat Sci Eng C-Mater, 42 (2014) 22-30. [20] A. Larrañaga, D. Ramos, H. Amestoy, E. Zuza, J.R. Sarasua, Coating of bioactive glass particles with mussel-inspired polydopamine as a strategy to improve the thermal stability of poly(L-lactide)/bioactive glass composites, Rsc Adv, 5 (2015) 65618-65626. [21] J. Shen, D. Shi, C. Shi, X. Li, M. Chen, Fabrication of dopamine modified polylactide-poly(ethylene glycol) scaffolds with adjustable properties, J Biomat Sci-Polym E, 28 (2017) 2006. [22] Z. Min, K. Li, Y. Zhu, J. Zhang, X. Ye, 3D-printed hierarchical scaffold for localized isoniazid/rifampin drug delivery and osteoarticular tuberculosis therapy, Acta Biomater, 16 (2015) 145. [23] C. Wu, W. Fan, J. Chang, Y. Xiao, Mussel-inspired porous SiO2 scaffolds with improved mineralization and cytocompatibility for drug delivery and bone tissue engineering, J Mater Chem, 21 (2011) 18300-18307. [24] S. Zhao, J. Zhang, M. Zhu, Y. Zhang, Z. Liu, Y. Ma, Y. Zhu, C. Zhang, Effects of functional groups on the structure, physicochemical and biological properties of mesoporous bioactive glass scaffolds, J Mater Chem B, 3 (2015) 1612-1623.
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[25] P.Y. Wang, L. Clements, H. Thissen, W.B. Tsai, N. Voelcker, High-throughput characterisation of osteogenic differentiation of human mesenchymal stem cells using pore size gradients on porous alumina, Biomater Sci-UK, 1 (2013) 924-932. [26] M. Shamsi, M. Karimi, M. Ghollasi, N. Nezafati, M. Shahrousvand, M. Kamali, A. Salimi, In vitro proliferation and differentiation of human bone marrow mesenchymal stem cells into osteoblasts on nanocomposite scaffolds based on bioactive glass (64SiO2-31CaO-5P2O5)-poly-l-lactic acid nanofibers fabricated by electrospinning method, J Mat Sci Eng C-Mater, 78 (2017) 114. [27] F.O. Obiweluozor, A. Ghavaminejad, B. Maharjan, J. Kim, H.P. Chan, C.S. Kim, A mussel inspired self-expandable tubular hydrogel with shape memory under NIR for potential biomedical applications, J Mater Chem B, 5 (2017). [28] H. Liu, W. Li, W. Wen, B. Luo, M. Liu, S. Ding, C. Zhou, Mechanical properties and osteogenic activity of poly(l-lactide) fibrous membrane synergistically enhanced by chitosan nanofibers and polydopamine layer, J Mat Sci Eng C-Mater, 81 (2017) 280. [29] H. Wang, C. Wu, X. Liu, J. Sun, G. Xia, W. Huang, R. Song, Enhanced mechanical and thermal properties of poly( l -lactide) nanocomposites assisted by polydopamine-coated multiwalled carbon nanotubes, Colloid Polym Sci, 292 (2014) 2949-2957. [30] H. Wang, G. Xie, M. Fang, Z. Ying, Y. Tong, Y. Zeng, Mechanical reinforcement of graphene/poly(vinyl chloride) composites prepared by combining the in-situ suspension polymerization and melt-mixing methods, Compos Part B-Eng, 113 (2017) 278-284. [31] C. Shuai, P. Feng, P. Wu, Y. Liu, X. Liu, D. Lai, C. Gao, S. Peng, A combined nanostructure constructed by graphene and boron nitride nanotubes reinforces ceramic scaffolds, Chem Eng J, (2016). [32] Y. Xu, D. Gao, P. Feng, C. Gao, S. Peng, H.T. Ma, S. Yang, C. Shuai, A mesoporous silica composite scaffold: Cell behaviors, biomineralization and mechanical properties, Appl Sur Sci, 423 (2017). [33] P. Feng, S. Peng, P. Wu, C. Gao, W. Huang, Y. Deng, T. Xiao, C. Shuai, A nano-sandwich construct built with graphene nanosheets and carbon nanotubes enhances mechanical properties of hydroxyapatite–polyetheretherketone scaffolds, Int J Nanomed, 11 (2016) 3487-3500. [34] Pei Feng, Ping Wu, Chengde Gao, Youwen Yang, Wang Guo, Wenjing Yang, Cijun Shuai. A multi-material scaffold owning tunable properties: towards bone tissue repair. Advanced Science, 2018, 1700817, 1-15 [35] R. Jeyakumar, P.S. Sampath, R. Ramamoorthi, T. Ramakrishnan, Structural, morphological and mechanical behaviour of glass fibre reinforced epoxy nanoclay composites, Int J Adv Manuf Tech, (2017) 1-9. [36] G. He, Z. Yang, L. Pan, J. Zhang, S. Liu, Q.L. Yan, Bioinspired Interfacial Reinforcement of Polymer-based Energetic Composites with High Loading of Solid Explosive Crystals, J Mater Chem A, 5 (2017). [37] Z.X. Wang, C.H. Lau, N.Q. Zhang, Y.P. Bai, L. Shao, Mussel-inspired tailoring of membrane wettability for harsh water treatment, J Mater Chem A, 3 (2015) 2650-2657. [38] E.V. Todorova, G.E. Chernev, S.P. Djambazov, Synthesis and characterization of silica hybrid materials applicable for defect remediation of concrete, Bulg Chem Commun, 47 (2015) 268-275. [39] M. Vukomanović, I. Sarčev, B. Petronijević, S.D. Skapin, N. Ignjatović, D. Uskoković, Poly(D,L-lactide-co-glycolide)/hydroxyapatite core-shell nanospheres. Part 4: a change of the
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surface properties during degradation process and the corresponding in vitro cellular response, Colloid Surface B, 91 (2012) 144-153. [40] Liu T, Wu P, Gao C, et al. Synergistic Effect of Carbon Nanotubes and Graphene on Diopside Scaffolds[J]. Biomed Res Int, 2016, 2016:1-8. [41] N.G. Rim, S.J. Kim, Y.M. Shin, I. Jun, D.W. Lim, J.H. Park, H. Shin, Mussel-inspired surface modification of poly(L-lactide) electrospun fibers for modulation of osteogenic differentiation of human mesenchymal stem cells, Colloid Surface B, 91 (2012) 189. [42] D. Lozano, M.J. Feito, S. Portal-Núñez, R.M. Lozano, M.C. Matesanz, M.C. Serrano, M. Vallet-Regí, M.T. Portolés, P. Esbrit, Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells, Acta Biomater. 8 (2012) 2770.
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Fig. 1 Micromorphology of (a) MBG and (b) p-MBG, the illustrations were optical photographs of p-MBG and MBG powder. Embossments on p-MBG may be due to polydopamine attachment. (c) Schematic diagram of hydrogen bonding between polydopamine and p-MBG. (d) FTIR, (e) XPS survey spectra and (f) TG curves of -1
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p-MBG and MBG. The peak at 1506 cm was corresponded to the v(C=N) stretching
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and N-H bending vibration mode of aromatic rings in polydopamine, which suggested the successful adhesion of polydopamine. The N1s peak appearing at 400 eV was attributed to the amine groups of polydopamine, which further confirmed the success of polydopamine modification of p-MBG.
Fig. 2 N2 adsorption-desorption isotherm (a) and mesopore size distribution (b) of
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p-MBG and MBG. It indicated that polydopamine modification had no obvious influence on the specific surface area and size distribution.
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Fig. 3 Elliptic cylinder scaffolds (12×9×8 mm): (a) PLLA/p-MBG scaffold; (b) PLLA/MBG. The scaffold has an interconnected porous structure, and the size of struts and pores was about 400 μm.
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Fig. 4 (a) FTIR spectra of the PLLA/p-MBG, PLLA/MBG and PLLA scaffolds. The shift of the peak at 1753 cm
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to lower wavenumber indicated the intermolecular
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interaction between p-MBG and PLLA. (b) Schematic of the possible interactions between p-MBG and PLLA matrix. Hydrogen bonding between catechol groups of p-MBG and ester groups of PLLA established a cross-linked bridge, which facilitates the enhancement of interfacial bonding.
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Fig. 5 The micromorphology of PLLA/MBG (a-c) and PLLA/p-MBG (d-f) scaffold with various contents of MBG and p-MBG: (a, d) 5 wt. %, (b, e) 10 wt. % and (c, f) 15 wt. %. p-MBG exhibited uniform dispersion in PLLA matrix compared to MBG. Filler effects on compressive strength (g) and modulus (h) of scaffolds. The introduction of p-MBG and MBG significantly improved the compressive strength and modulus of PLLA scaffold. Moreover, PLLA/p-MBG scaffold had higher compressive strength and modulus than PLLA/MBG scaffold with the same filler contents. Especially when the filler content was 15 wt. %, the mechanical strengthening effect of p-MBG was more significant.
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Fig. 6 Water contact angle of scaffolds (the mean ± SD is n = 5). The addition of p-MBG and MBG significantly improved the hydrophilic properties of scaffold. PLLA/p-MBG scaffold showed lower water contact angle at the same filler loading compared to PLLA/MBG scaffold, indicating that polydopamine modification improved hydrophilicity.
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Fig. 7 MG 63 cells after 3 days culture on PLLA/MBG (a-c) and PLLA/p-MBG (d-f) scaffold with MBG and p-MBG: (a, d) 5 wt. %, (b, e) 10 wt. % and (c, f) 15 wt. %. The number of cells and spreading area on PLLA/p-MBG scaffold was significantly better than those on PLLA/MBG scaffold.
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Fig. 8 Viability (A) and ALP activity (B) analysis of MG63 cells after cultivated on PLLA/MBG (1-3) and PLLA/p-MBG (4-6) scaffold with MBG and p-MBG: (1, 4) 5 wt. %, (2, 5) 10 wt. % and (3, 6) 15 wt. %. PLLA scaffold served as control group. The results indicated that PLLA/p-MBG scaffold had good cytocompatibility.
Table 1 Mesoporous structure parameters of p-MBG and MBG
2 -1
3 -1
SBET/m g
VP/cm g
DP/nm
p-MBG
396
0.541
5.487
MBG
577
0.668
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Samples
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S0927-7765(18)30365-5 https://doi.org/10.1016/j.colsurfb.2018.05.065 COLSUB 9384
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
6-2-2018 28-5-2018 29-5-2018
Please cite this article as: Yong Xu, Ping Wu, Pei Feng, Wang Guo, Wenjing Yang, Cijun Shuai, Interfacial reinforcement in a poly-l-lactic acid/mesoporous bioactive glass scaffold via polydopamine, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.05.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interfacial reinforcement in a poly-l-lactic acid/mesoporous bioactive glass scaffold via polydopamine Yong Xu 1,2,#, Ping Wu 3,#, Pei Feng 1, Wang Guo 1, Wenjing Yang 1, Cijun Shuai 1,4,5,* 1 State Key Laboratory of High Performance Complex Manufacturing, College of Mechanical and Electrical Engineering, Central South University, Changsha 410083, China
China 3 College of Chemistry, Xiangtan University, Xiangtan 411105, China
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4 Jiangxi University of Science and Technology, Ganzhou 341000, China
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2 Department of Mechanical and Energy Engineering, Shaoyang University, Shaoyang 422004,
5 Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha 410008, China
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# These authors contributed equally to this work
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* Correspondence: [email protected]; Tel: +86-731-84805412; Fax: +86-731-88879044
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Graphical abstract
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Caption: Graphical abstract demonstrated the preparation and performance tests of poly-l-lactic acid (PLLA)-based scaffolds. Polydopamine modified mesoporous bioactive glass (p-MBG) was prepared by oxidation self-polymerization of dopamine in a tris(hydroxymethyl) aminomethane hydrochloride buffer solution (Tris-HCL). Scaffolds were prepared by selective laser sintering process (SLS).
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Highlights
PLLA/p-MBG scaffold was constructed by selective laser sintering.
Polydopamine was used as a cross-linking bridge to improve interfacial interaction.
PLLA/p-MBG scaffold has improved mechanical properties and hydrophilicity.
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PLLA/p-MBG scaffold also has good biocompatibility.
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Abstract
The weak interfacial interaction between polymer and bioceramic leads to a low reinforcing
effect, even though polymer/bioceramic scaffolds are considered potential implants. In this work, a porous poly-l-lactic acid (PLLA) scaffold containing polydopamine-modified mesoporous bioactive glass (p-MBG) was fabricated by selective laser sintering. The polydopamine was introduced as a cross-linking bridge between MBG and PLLA to improve the interfacial
interaction between of them. On the one hand, the amine groups of polydopamine formed strong hydrogen bonds with the hydroxyl groups of MBG. On the other hand, the catechol groups of polydopamine formed strong hydrogen bonds with the ester groups of PLLA. The PLLA/p-MBG scaffold showed higher compressive strength and modulus (62.9 MPa and 3.6 GPa, respectively) than the PLLA/MBG scaffold (41.9 MPa and 2.1 GPa, respectively). This result was explained by the fact that the enhanced interfacial adhesion promoted the dispersion of p-MBG and increased
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the efficiency of stress transfer in the matrix. Additionally, the PLLA/p-MBG scaffold showed good cytocompatibility. This study suggested that the PLLA/p-MBG scaffold may have a potential
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application in tissue engineering.
Keywords: cross-linking bridge; interfacial reinforcement; cytocompatibility; mesoporous bioactive glass; polydopamine
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1 Introduction
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Incorporating bioceramic into a polymer scaffold to improve the biological and mechanical
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properties has attracted a considerable amount of attention. Nevertheless, the mechanical properties of the scaffold were decreased at high filler loading due to the weak interfacial
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interaction between the bioceramic and polymer resulting from their significant difference in
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physicochemical properties [1-3]. The weak interfacial interactions result in a morphological defect or failure at the interface, causing deterioration of the mechanical properties of the scaffold [4, 5]. Therefore, it is of great importance to enhance the interfacial interaction between the
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polymer and bioceramic. Several researchers have been committed to the enhancement of the interfacial interaction between bioceramics and polymers [6-8]. As reported by Rezabeigi E et al.,
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45S5 bioactive glass modified by 3-methacryloxypropyltriethoxysilane exhibited good dispersion in polylactic acid, and the polylactic acid/bioglass scaffold had improved mechanical properties [9]. Fang Z et al. reported that using γ-aminopropyltriethoxysilane to modify hydroxyapatite (HA)
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could improve the dispersion and interfacial compatibility of HA in poly-l-lactic acid (PLLA), which improved the mechanical properties of the HA/PLLA scaffold [10]. Nevertheless, these methods might have potential side effects due to the use of toxic chemicals. Therefore, it is necessary to develop a safe and effective modification strategy for improving the interfacial interaction between the bioceramics and polymers. Dopamine (C8H11O2N, 4-(2-aminoethyl) benzene-1,2-diol) is a type of cell signal agent widely
found in organisms. It can self-polymerize to form polydopamine, which has a similar structure to the protein melanin [11]. Recently, the application of polydopamine in tissue engineering has drawn keen attention due to its excellent biocompatibility and biodegradability [12, 13]. More importantly, polydopamine can form non-covalent crosslinks with matrices that contain ester and hydroxyl groups [14]. PLLA is a type of polyester polymer with good biocompatibility. The ester groups in its molecular chain may form strong hydrogen bonds with the catechol groups of
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polydopamine. Mesoporous bioactive glass (MBG) is a newly developed bioactive material. The hydroxyl groups enriched on its mesoporous channels also may form strong hydrogen bonds with
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the amine groups of polydopamine [15]. Therefore, polydopamine is expected to establish a cross-linking bridge between PLLA and MBG, allowing the mechanical properties of the scaffold to be improved while retaining its good biological properties.
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In this work, to address the weak interfacial interactions between mesoporous bioactive glass
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(MBG) and poly-l-lactic acid (PLLA), polydopamine was introduced as a cross-linking bridge
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between of them. The polydopamine-modified mesoporous bioactive glass (p-MBG) was prepared based on the self-polymerization of dopamine and the mesoporous structure was analyzed.
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Moreover, a porous PLLA scaffold containing p-MBG was fabricated by selective laser sintering.
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The compressive strength and modulus of scaffold were measured. The micromorphology of p-MBG and its dispersion in scaffold were investigated. In addition, the mechanisms of interfacial interaction between p-MBG and PLLA were analyzed. Besides, the cytocompatibility and
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hydrophilicity of scaffold were also evaluated.
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2 Materials and methods
2.1 Materials and preparation Poly-l-lactic acid (PLLA, Mn 150,000; inherent viscosity 2.5~3.0 dl/g in chloroform at 25℃)
was kindly provided by Shenzhen Polymtek Biomaterial Co., Ltd. (Shenzhen, China). Dopamine
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hydrochloride and tris(hydroxymethyl) aminomethane (Tris, 99%) were purchased from Sigma and used as received. MBG was prepared by a sol-gel method process, according to the research reported by Chang J et al. [16]. The modification of MBG was performed by the oxidative polymerization of dopamine under alkaline conditions. Briefly, 4 g of MBG was dispersed in 1000 mL Tris-HCl buffer solution (10 mM, pH 8.5) and treated ultrasonically for 30 min. Subsequently, 2g of dopamine hydrochloride was added to the solution and mildly stirred for 5h at ambient
temperature. Then, the solution was filtrated and rinsed with deionized water 3 times to remove the polydopamine that was not adhered on the MBG. Finally, a dark brown p-MBG powder was obtained after drying overnight at 60 °C. To prepare the blend powder, a predetermined amount of p-MBG or MBG was separately added to beakers containing 50 ml of absolute ethanol and then sonicated for 30 min. Subsequently, an appropriate amount of PLLA was added to the suspension and treated with ultrasonic vibration and magnetic stirring for another 30 min. The suspension was
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then filtered, dried at 60 °C for 12 h and mechanically ground to obtain a mixed powder.
The scaffold was fabricated via selective laser sintering. During the preparation, the laser
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beam selectively scanned the powder on the sintering platform according to the slice profile of the scaffold. The laser energy made the temperature of the powder rise sharply to the melting point, which caused the powder to melt and rapidly solidify. Subsequently, the
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sintering platform was moved down a powder layer thickness and the sintering process was
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repeated. The process parameters remained constant throughout the sintering: laser power of
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2.3 W, spot diameter of 50 μm, scan rate of 100 mm/s, scan line spacing of 1 mm and powder layer thickness of 0.1 mm. After the sintering was completed, compressed air was used to
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remove the unsintered powder and clean the scaffold.
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2.2 Characterization
The micromorphology of p-MBG was characterized by scanning electron microscopy (SEM, Tescan Mira3 Lmu, Co., Czechia). N2 adsorption–desorption isotherms were obtained on a
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Beshide 3H-2000PS2 at 200 °C under continuous adsorption conditions. The surface area and pore size distribution of p-MBG powder were analyzed by the Brunauer-Emmett-Teller (BET) and
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Barrett-Joyner-Halenda (BJH) method. The chemical composition of p-MBG was characterized by Fourier transform infrared spectrophotometer (FTIR, Thermo Scientific Co., Madison, WI, USA) and X-ray photoelectron spectroscopy (XPS, ThermoFisher-VG Scientific, USA). For
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thermo-gravimetric (TG) analysis, 8-10 mg of sample powder was placed in ceramic crucible and heated from environment temperature to 800 °C within a TG analyzer (Nanjin Dazhan Institute of Electromechanical Technology STA-200, China), at a heating rate of 20 °C/min in nitrogen condition. The micromorphology of the scaffold was characterized via SEM to analyze the distribution of p-MBG. The compressive strength and modulus of the scaffold were tested using a universal
mechanical testing machine (Shanghai Zhuoji Instruments Co., Ltd., Shanghai, China) at a cross-head speed of 0.5 mm/min. Each test consisted of five samples for replicates, from which the average value and standard deviation were calculated. The chemical groups of the PLLA/p-MBG scaffold were observed by FTIR to examine the interaction between p-MBG and PLLA. The hydrophilicity of these scaffolds was evaluated via water contact angle measurement with an Attension Theta system (Biolin Scientific Co. Ltd., Stockholm, Sweden). For each sample,
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five measurements were performed, from which the average value and standard deviation were calculated.
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2.3 Cell culture
MG-63 cells (Cellular Biology Institute, Shanghai, China) were selected in cytocompatibility analysis. Disk scaffold samples (10 mm in diameter and 2 mm in height) were used to evaluate the
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effect of the scaffold material composition on cell behavior (adhesion and viability). Cells were
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cultured in DMEM (KeyGEN BioTECH) supplemented with 10% fetal bovine serum (FBS) and 1%
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penicillin/streptomycin sulfate at a density of 1 × 105 cells/scaffold under a humidified atmosphere of 95% air. The culture medium was renewed every two days. All the instruments and scaffold
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used in this experiment were previously UV sterilized for one hour.
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After 3 days of culturing, the cell/scaffold complexes were collected from the culture medium and rinsed with phosphate buffered solution (PBS) for three times. Subsequently, the cell/scaffold complexes were fixed with 3% glutaraldehyde for 30 min and dehydrated with gradient ethanol.
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Finally, the cell/scaffold complexes were dried in air and subjected to gold sputtering to observe the cell morphology under SEM. The viability of the cells was analyzed by staining the viable
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cells and dead cells with calcein AM and propidium iodide (PI), respectively. Briefly, the cell/scaffold complexes were collected from the culture medium and rinsed twice with PBS. Then, cells were removed from the cell/scaffold complexes with trypsin solution and reseeded onto glass
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slides. After 12 h, cells were then incubated in a medium containing 2 μM calcein AM and 4 μM PI for 30 minutes at 37℃. Afterward, optical analysis was performed by a fluorescence microscope fitted with a digital camera. After osteoblast induction culture for 3 days, LabAssay (TM) ALP kit (Wako, Osaka, Japan) was used to detect the level of alkaline phosphatase (ALP) to evaluate osteogenic differentiation of MG-63 cells. Specifically, a 0.25% trypsin solution was used to remove adherent cells. After
then, cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS, followed by ALP staining performed according to the manufacturer's instructions. Finally, images of stained cells were taken by an inverted microscope (TE2000U, Nikon, Japan). 2.4 Statistical analysis The data of experiments were expressed as the mean ± standard deviation and analyzed using Levene’s test. Statistically significant differences between groups were measured using the
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Student-Newman-Keuls post-hoc test (p). The difference was regarded as statistically significant when * p <0.05.
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3 Results and discussions 3.1 Polydopamine modified MBG
The micromorphology and structure schematic of p-MBG are shown in Fig. 1. The color of
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MBG powder changed from white to dark brown after modification, as shown in the inset of Fig.
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1a and b, indicating that polydopamine successfully formed on MBG. In this process, the amine
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groups of dopamine formed hydrogen bonds with the hydroxyl groups of MBG, and then underwent oxidative self-polymerization to form polydopamine on the surface of MBG [17]. As
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observed from the micromorphology in Fig. 1b, some embossments that existed on p-MBG
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surface are potentially due to the coverage of polydopamine. A schematic diagram of hydrogen bonding between polydopamine and p-MBG is shown in Fig. 1c. FTIR was used to analyze the surface chemical composition of powder, as presented in Fig. 1d.
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Absorption peaks appeared at approximately 3500 and 1637 cm-1, which were attributed to the asymmetric stretching vibration band and bending vibration mode of the O-H of absorbed water,
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respectively [18]. The peaks appearing at 1100 and 802 cm-1 were corresponded to the asymmetric stretching vibration band of Si-O-Si and symmetrical stretching vibration band of Si-O, respectively [19]. For p-MBG, the peak at around 3500 cm-1, which may be due to the v(N-H)
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stretching mode of polydopamine, intensified compared to the MBG [20]. In addition, after modification, the peak at 802 cm-1 weakened and a new absorption peak was detected at 1506 cm-1, which may be due to the effects of v(C=N) stretching and N-H bending vibration mode of aromatic rings in polydopamine, respectively [21]. These results further demonstrated that polydopamine successfully formed on the MBG. Wide scan x-ray photoelectron spectroscopy (XPS) of p-MBG particles are shown in Fig. 1 e.
After polydopamine modification, the XPS spectra of p-MBG particles displayed a new peak at 400 eV, which corresponds to N1s. The presence of nitrogen was attributed to amine groups of polydopamine on the surface of p-MBG particles. Thermo-gravimetric (TG) analysis was used to estimate the amount of polydopamine attached on the p-MBG (Fig. 1f). The mass loss in the range of 50-200 °C was attributed to the adsorption of moisture from environment. And the mass loss at temperatures over 200 °C was caused by the decomposition of polydopamine. By calculating the
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amount of coke in samples above 650 °C, it was confirmed that the amount of polydopamine attached on p-MBG was about 12 wt. %.
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The N2 sorption isotherm and corresponding mesopore size distribution of p-MBG are illustrated in Fig. 2. The results indicated that polydopamine modification had no obvious influence on the mesopore structure and size distribution. As shown in Fig. 2a, p-MBG exhibited
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type IV isotherms and the capillary condensation increased from 0.4 to 0.6 of P/P0, which was
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representative of mesopores with relatively narrow pore size distribution [22]. The distribution of
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the corresponding BJH mesopore size in Fig. 2b was obtained from the adsorption branch. p-MBG showed slightly narrow pore size distribution, and the peak pore diameter of p-MBG was 5.487
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nm. In addition, the BET surface area of p-MBG was 396 m2g-1 and the BJH pore volume was
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reduced to 0.541 cm3g-1, as illustrated in Table 1. It was attributed to the occupation of polydopamine on the inner wall of mesopores as well as the disturbation of amidogen groups [23,
3.2 Scaffold
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24]. In conclusion, the mesoporous structure of p-MBG was well preserved.
Elliptical cylindrical scaffolds are exhibited in Fig. 3. Their dimensions were 12×9×8 mm
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(major axis × minor axis × height). Moreover, both the sizes of struts and pores were about 400μm, which is believed to favor the growth and vascularization of new bone [25]. The interconnected porous scaffold could carry out the transport of nutrients and provide physical support for the
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growth of new tissues. This result suggested that the selective laser sintering was an effective strategy for the preparation of bone repair scaffold. FTIR spectra of PLLA, PLLA/p-MBG and PLLA/MBG scaffolds are shown in Fig. 4a. The strong peak at 1758 cm-1 corresponded to the C=O stretching vibration of the ester group in PLLA [26]. For PLLA/p-MBG scaffold, this strong absorption peak shifted toward a lower wavenumber at 1753 cm-1, suggesting an intermolecular interaction between p-MBG and PLLA [27]. This shift
was ascribed to the hydrogen bond formed between catechol groups of p-MBG and ester groups of PLLA [28]. Moreover, a simple hydrogen bond cross-linking sketch diagram is displayed in Fig. 4b, in which polydopamine acts as a cross-linking bridge to connect p-MBG and PLLA. On the one hand, amine groups of polydopamine formed strong hydrogen bonds with the hydroxyl groups of MBG. On the other hand, catechol groups of polydopamine also formed strong hydrogen bonds with the ester groups of PLLA. Polydopamine cross-linking bridge enhanced the interface bonding
scaffold.
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3.3 The micromorphology and mechanical properties of scaffold
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between p-MBG and PLLA, thereby potentially improving the mechanical properties of the
The dispersion of p-MBG and MBG in the scaffold is illustrated in Fig. 5. EDS spectra at the p1 and p2 sites are provided as illustrations in Fig. 5c and f. The emergence of peaks of Si and N
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indicated that these white particles are MBG and p-MBG, respectively. When the content of
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addition was 5 wt. %, both p-MBG and MBG showed uniform dispersion in the PLLA matrix, as
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shown in Fig. 5a and d. However, for PLLA/MBG scaffold, an agglomeration phenomenon (as shown with red arrows) began to occur and became conspicuous as the content increased from 10
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to 15 wt. % (Fig. 5b and c). Comparatively, the dispersion of p-MBG in the PLLA matrix was
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much improved at the same loading levels (Fig. 5e and f). This was because hydrogen bonding formed between the catechol group of p-MBG and the ester group of PLLA, as schematically shown in Fig. 4b, which promoted the dispersion of p-MBG in the matrix [29]. The uniform
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dispersion of the filler in the matrix might be helpful to improve the ultimate mechanical and other properties of the scaffold [30, 31].
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The dependence of the compressive strength and modulus of PLLA/p-MBG and PLLA/MBG
scaffold on filler contents is shown in Fig. 5g and h. Clearly, the introduction of p-MBG and MBG significantly improved the compressive strength and modulus of PLLA scaffold. The compressive
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strength and modulus of PLLA scaffold were 20.8 ± 2.5 MPa and 1.8 ± 0.1 GPa, respectively.
For PLLA/MBG scaffold, the compressive strength and modulus first increased and then decreased as the MBG content increased. The maximum compressive strength and modulus of the PLLA/MBG scaffold were 50.2 ± 4.3 MPa and 3.1 ± 0.2 GPa, respectively, at 10 wt. %. It was attributed to the fact that the high strength and modulus of MBG and the particle enhancement effect had a positive effect on increasing the compressive strength and modulus of the scaffold [32,
33]. Additionally, when the content reached 15 wt. %, the decrease in strength and modulus was caused by the aggregation of MBG in the matrix [34]. For PLLA/p-MBG scaffold, the compressive strength and modulus increased with increasing p-MBG contents. In detail, the compressive strength and modulus increased from 45.3 ± 2.2MPa and 2.3 ± 0.2GPa to 62.9 ± 5.2 MPa and 3.6 ± 0.3 GPa, respectively, as the p-MBG content increased from 5 to 15 wt. % (Fig. 5g and h). In comparison, PLLA/p-MBG scaffold had a higher
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compressive strength and modulus than PLLA/MBG scaffold with the same filler contents. This finding could be explained by the following two aspects. On the one hand, the homogeneous
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dispersion of p-MBG in the PLLA matrix prevented agglomeration, resulting in significant
particle reinforcement [35]. On the other hand, polydopamine acted as a cross-linking bridge between PLLA and p-MBG, which promoted the effective stress transfer at the interface [36].
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3.4 The hydrophilicity of scaffold
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The water contact angle of PLLA/p-MBG scaffold is shown in Fig. 6. Obviously, PLLA
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scaffold exhibited hydrophobic properties with a water contact angle of 105.8 ± 6.4°. The water contact angle of PLLA/MBG scaffold decreased from 82.5 ± 5.3° to 51.6 ± 2.5° as the MBG
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content increased from 5 to 15 wt. %, exhibiting hydrophilic properties. This change indicated that
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the addition of MBG significantly improved the hydrophilicity of PLLA scaffold. Comparatively, PLLA/p-MBG scaffold showed a lower water contact angle at the same loading level, indicating that polydopamine modification could further improve hydrophilicity. There were two probable
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factors responsible for the improved hydrophilicity. On the one hand, even distribution of p-MBG led to an increase in the contact surface area, and hence improved the hydrophilicity of the
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scaffold [37]. On the other hand, polydopamine contained a large amount of hydrophilic groups, such as catechol and amine groups, which might be of benefit to further improve the hydrophilicity [38]. The improved hydrophilicity might be part of the potential contributors to the
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stimulation of cellular response [5]. 3.5 Cell culture Adherent cell morphology after culture for 3 days is shown in Fig. 7. It can be seen that the cells spreading area increased with increasing filler content. Cells were spindle-shaped when the addition content was 5 wt. %, as shown in Fig. 7a and d. Cells extended to a wider range of the scaffold sample surface (Fig. 7b and e) at a filler loading of 10 wt. %. When p-MBG increased up
to 15 wt. %, the adherent cells were fully stretched and diffused along the scaffold (Fig. 7f). In general, cells on PLLA/p-MBG scaffold samples exhibited good attachment morphology, which suggested that these scaffold material were suitable for cell adhesion. There were likely two probable factors that enhanced cell adhesion. On the one hand, polydopamine itself served as an anchor between cell and matrix due to the hydrophilicity and strong adhesion [39]. On the other hand, the uniform dispersion of p-MBG in the scaffold could exhibit more contact surface area to
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provide more attachment sites for cell attachment [40].
The viability of MG-63 cells was assessed by immunofluorescence. After 3 days of culture,
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cells were removed from the cell/scaffold complexes with trypsin solution and reseeded onto glass slides to observe cell morphology, as shown in Fig. 8A. The cells showed good diffusion
morphology and the cell density increased with increasing p-MBG content, indicating that the
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PLLA/p-MBG scaffold material had good cytocompatibility. The cytocompatibility of the scaffold
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material was attributed to the fact that the large specific surface area of the mesoporous structure
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provided cells with numerous adhesion sites. In addition, the OH- and NH2+ groups of polydopamine could improve the hydrophilicity and adjust the surface charge of the scaffold,
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which provided opportunities for the adhesion and proliferation of cells [41]. The ALP activity is
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an early feature of osteoblast differentiation and plays a key role in early bone formation. Hence the cell ALP activity after 3 days of culture was also evaluated and is shown in Fig.8B. It can be seen that the ALP activity of cells increased with the increasing filler loading, suggesting that
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PLLA/p-MBG scaffold material had the ability to stimulate cell differentiation. This was mainly due to the release of active elements (silicon and calcium) from the composite scaffold, which can
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stimulate cell gene expression and mineralization and further induce osteoblast differentiation [42].
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4 Conclusions
In this work, three-dimensional porous PLLA/p-MBG scaffolds for tissue engineering implants
were constructed via selective laser sintering process. Polydopamine was used as the interface compatibilizer to improve the interfacial interaction between MBG and PLLA. BET and BJH analysis showed that the mesoporous structure of p-MBG was well preserved. Polydopamine, as a cross-linking bridge between PLLA and MBG, greatly improved the interfacial compatibility and mechanical properties of PLLA/p-MBG scaffold. Cell culture results indicated that cells
preferentially adhered, proliferated and differentiated on PLLA/p-MBG scaffold, indicating that these scaffold material had good biocompatibility. The results suggest that PLLA/p-MBG scaffold may be a potential candidate for tissue engineering implant applications. Acknowledgements This work was supported by the following funds: (1) The Natural Science Foundation of China (51575537, 81572577, 51705540); (2) Hunan Provincial Natural Science Foundation of China
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(2016JJ1027); (3) The Project of Innovation-driven Plan of Central South University
(2016CX023); (4) The Open-End Fund for the Valuable and Precision Instruments of Central
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South University; (5) The fund of the State Key Laboratory of Solidification Processing in NWPU
(SKLSP201605); (6) National Postdoctoral Program for Innovative Talents (BX201700291); (7) The Project of State Key Laboratory of High Performance Complex Manufacturing, Central South
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University; (8) The Project of Hunan Provincial Science and Technology Plan (2017RS3008); (9)
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The Fundamental Research Funds for the Central Universities of Central South University
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(2018ZZTS145). References
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surface properties during degradation process and the corresponding in vitro cellular response, Colloid Surface B, 91 (2012) 144-153. [40] Liu T, Wu P, Gao C, et al. Synergistic Effect of Carbon Nanotubes and Graphene on Diopside Scaffolds[J]. Biomed Res Int, 2016, 2016:1-8. [41] N.G. Rim, S.J. Kim, Y.M. Shin, I. Jun, D.W. Lim, J.H. Park, H. Shin, Mussel-inspired surface modification of poly(L-lactide) electrospun fibers for modulation of osteogenic differentiation of human mesenchymal stem cells, Colloid Surface B, 91 (2012) 189. [42] D. Lozano, M.J. Feito, S. Portal-Núñez, R.M. Lozano, M.C. Matesanz, M.C. Serrano, M. Vallet-Regí, M.T. Portolés, P. Esbrit, Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells, Acta Biomater. 8 (2012) 2770.
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Fig. 1 Micromorphology of (a) MBG and (b) p-MBG, the illustrations were optical photographs of p-MBG and MBG powder. Embossments on p-MBG may be due to polydopamine attachment. (c) Schematic diagram of hydrogen bonding between polydopamine and p-MBG. (d) FTIR, (e) XPS survey spectra and (f) TG curves of -1
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p-MBG and MBG. The peak at 1506 cm was corresponded to the v(C=N) stretching
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and N-H bending vibration mode of aromatic rings in polydopamine, which suggested the successful adhesion of polydopamine. The N1s peak appearing at 400 eV was attributed to the amine groups of polydopamine, which further confirmed the success of polydopamine modification of p-MBG.
Fig. 2 N2 adsorption-desorption isotherm (a) and mesopore size distribution (b) of
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p-MBG and MBG. It indicated that polydopamine modification had no obvious influence on the specific surface area and size distribution.
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Fig. 3 Elliptic cylinder scaffolds (12×9×8 mm): (a) PLLA/p-MBG scaffold; (b) PLLA/MBG. The scaffold has an interconnected porous structure, and the size of struts and pores was about 400 μm.
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Fig. 4 (a) FTIR spectra of the PLLA/p-MBG, PLLA/MBG and PLLA scaffolds. The shift of the peak at 1753 cm
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to lower wavenumber indicated the intermolecular
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interaction between p-MBG and PLLA. (b) Schematic of the possible interactions between p-MBG and PLLA matrix. Hydrogen bonding between catechol groups of p-MBG and ester groups of PLLA established a cross-linked bridge, which facilitates the enhancement of interfacial bonding.
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Fig. 5 The micromorphology of PLLA/MBG (a-c) and PLLA/p-MBG (d-f) scaffold with various contents of MBG and p-MBG: (a, d) 5 wt. %, (b, e) 10 wt. % and (c, f) 15 wt. %. p-MBG exhibited uniform dispersion in PLLA matrix compared to MBG. Filler effects on compressive strength (g) and modulus (h) of scaffolds. The introduction of p-MBG and MBG significantly improved the compressive strength and modulus of PLLA scaffold. Moreover, PLLA/p-MBG scaffold had higher compressive strength and modulus than PLLA/MBG scaffold with the same filler contents. Especially when the filler content was 15 wt. %, the mechanical strengthening effect of p-MBG was more significant.
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Fig. 6 Water contact angle of scaffolds (the mean ± SD is n = 5). The addition of p-MBG and MBG significantly improved the hydrophilic properties of scaffold. PLLA/p-MBG scaffold showed lower water contact angle at the same filler loading compared to PLLA/MBG scaffold, indicating that polydopamine modification improved hydrophilicity.
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Fig. 7 MG 63 cells after 3 days culture on PLLA/MBG (a-c) and PLLA/p-MBG (d-f) scaffold with MBG and p-MBG: (a, d) 5 wt. %, (b, e) 10 wt. % and (c, f) 15 wt. %. The number of cells and spreading area on PLLA/p-MBG scaffold was significantly better than those on PLLA/MBG scaffold.
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Fig. 8 Viability (A) and ALP activity (B) analysis of MG63 cells after cultivated on PLLA/MBG (1-3) and PLLA/p-MBG (4-6) scaffold with MBG and p-MBG: (1, 4) 5 wt. %, (2, 5) 10 wt. % and (3, 6) 15 wt. %. PLLA scaffold served as control group. The results indicated that PLLA/p-MBG scaffold had good cytocompatibility.
Table 1 Mesoporous structure parameters of p-MBG and MBG
2 -1
3 -1
SBET/m g
VP/cm g
DP/nm
p-MBG
396
0.541
5.487
MBG
577
0.668
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Samples
5.745