Article Cite This: ACS Biomater. Sci. Eng. 2018, 4, 1546−1557
Incorporation of Cu-Containing Bioactive Glass Nanoparticles in Gelatin-Coated Scaffolds Enhances Bioactivity and Osteogenic Activity Kai Zheng,†,‡ Jingjing Wu,†,§ Wei Li,⊥ Dirk Dippold,∥ Ying Wan,*,§ and Aldo R. Boccaccini*,‡
ACS Biomater. Sci. Eng. 2018.4:1546-1557. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/27/18. For personal use only.
‡
Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058, Germany § College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China ⊥ Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki FI-00014, Finland ∥ Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Martensstrasse 7, Erlangen 91058, Germany S Supporting Information *
ABSTRACT: Bioactive glass scaffolds (BGS) of 45S5 composition exhibit desired bioactivity, osteogenesis, and angiogenesis potential, being promising biomaterials for bone repair/regeneration. Natural polymer-based coatings, e.g., gelatin coating, are effective to enhance the mechanical properties of BGS. However, the presence of a coating may reduce the bioactivity and osteogenesis activity of the scaffolds. To address the issue of reduced osteogenic properties induced by polymer coatings, in this study, we incorporated Cucontaining bioactive glass nanoparticles (Cu-BGN: 95SiO22.5CaO-2.5CuO, in mol %), as bioactive fillers, into the gelatin coating. The bioactivity (apatite-forming ability) of the gelatin coated BGS was improved after the incorporation of Cu-BGN in the coating. Hydroxyapatite could form on the Cu-BGN/gelatin nanocomposite coated BGS within 1 day of immersion in simulated body fluid. The osteogenic activity as indicated by the ALP activity of MC3T3-E1 cells on the coated BGS was also significantly enhanced after the incorporation of Cu-BGN. In addition, the incorporation of Cu-BGN in the coating did not affect the highly porous and interconnected pore structure of BGS while the mechanical improvement induced by the gelatin coating remained after the addition of Cu-BGN. The attachment of MC3T3-E1 cells on the scaffolds was not influenced by the presence of Cu-BGN in the gelatin coating, while the cell proliferation was enhanced. In conclusion, the incorporation of bioactive nanoparticles into polymer coating is presented as a solution to the reduced bioactivity and osteogenic activity of polymer coated 45S5 BGS. The Cu-BGN/gelatin nanocomposite coated BGS exhibiting high bioactivity, appropriate mechanical properties, and osteogenic potential are candidate biomaterials for bone tissue engineering/regeneration. KEYWORDS: silicate bioactive glasses, porous scaffolds, bioactive nanoparticles, bone regeneration, copper
1. INTRODUCTION
Among numerous candidate materials, bioactive glasses (BGs) are particularly promising biomaterials for developing BTE scaffolds, considering their unique properties such as bonebonding ability and osteogenic induction.6,7 The physicochemical and biological behaviors of BGs are closely related to their composition. For example, the presence of copper in the composition at a proper concentration can significantly improve the angiogenesis activity of BGs.8 45S5 BG (45SiO2+24.5Na2O +24.5CaO+6P2O5, in wt %), the first developed BG, can bond to bone and induce bone growth.9 45S5 BG is also reported to be
Efficient repair of bone defects remains a challenge, particularly for large bone defects that lack the potential of spontaneous healing.1 Many efforts have been made to treat bone defects, such as using autografts and allografts. In the last decades, bone tissue engineering (BTE) has emerged as a promising strategy to address this challenge,2,3 in which scaffolds, osteogenic cells, and signaling biomolecules are usually combined to achieve successful bone regeneration/repair.2 In the BTE strategy, scaffolds play a pivotal role in supporting cell adhesion, growth, migration, and differentiation as well as inducing and guiding new tissue growth.4 These interactions between cells and scaffolds are regulated by many factors, such as the composition, porosity, and surface topography of the scaffolds.4,5 © 2018 American Chemical Society
Received: January 15, 2018 Accepted: April 19, 2018 Published: April 19, 2018 1546
DOI: 10.1021/acsbiomaterials.8b00051 ACS Biomater. Sci. Eng. 2018, 4, 1546−1557
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research, we selected the particles with a composition of 95SiO22.5CaO-2.5CuO (in mol %), as the inorganic filler, for the nanocomposite coating due to their advantages of high bioactivity and good dispersity for composite development.28,29 In addition, Cu-BGN have shown the capability of releasing Cu ions that can induce angiogenesis and antibacterial activity.29,30 A gelatin coating was capable of significantly improving the mechanical properties of 45S5 BG scaffolds, but it reduced the apatite-forming ability of the scaffolds.17 In this study, we investigated the influence of the presence of Cu-BGN in the gelatin coating on the mechanical properties, apatite-forming ability, cytotoxicity, and osteogenesis activity of 45S5 BG scaffolds. The results indicated that the addition of Cu-BGN did not affect the improvement in mechanical properties while significantly enhancing the apatite-forming ability that was comparable with that of the uncoated 45S5 BG scaffold. Additionally, it was investigated if the presence of Cu-BGN at proper concentrations could enhance the osteogenic activity of scaffolds.
able to facilitate vascularization and to be antibacterial at proper concentrations.6,7,10 45S5 BG can be fabricated into porous scaffolds for BTE, which can be achieved by using various techniques. In particular, 45S5 BG scaffolds developed by the foam replica method meet several significant criteria for an ideal BTE scaffold. For instance, these scaffolds have high porosity and a well-defined interconnected pore structure, which are beneficial for bone ingrowth.7,11 However, such scaffolds suffer from unsatisfactory mechanical properties because of their high porosity and limited densification behavior of 45S5 BG during sintering.11 Toward strengthening and toughening BG scaffolds, many strategies, such as tailoring scaffold morphology/structure and modifying the surface properties of scaffold struts, have been employed.12,13 For example, 3D printed BG scaffolds with predesigned structure may exhibit high mechanical properties that are suitable for load-bearing bone applications.14,15 However, a relatively low porosity and/or a pore structure with limited interconnection can be found in some scaffolds with high mechanical strength.13 The strategy of polymer coating thus emerges as an effective solution to improving the mechanical properties of 45S5 BG scaffolds without compromising their highly porous and interconnected pore structure.16 The polymer coating may also bring in novel functionalities to the scaffolds, e.g., antibacterial activity and drug delivery capability.17,18 Biodegradable and biocompatible polymers including synthetic19 and natural ones17 or their combination20 have been widely used to coat 45S5 BG scaffolds. These polymers can be coated on 45S5 BG scaffolds in the form of films/membranes20 or particles.21 Both forms are capable of leading to an improvement in mechanical properties. However, a continuous polymer coating may cause a reduction in bioactivity and/or biological activity of the scaffold.17,20 Gelatin, a water-soluble natural polymer, has shown its potential as a coating material for inorganic scaffolds.17,22 However, the apatite-forming ability of BG scaffolds may be weakened by the presence of gelatin coating.17 In addition, the ion exchange and release behaviors of BG scaffolds in body fluid may be influenced by polymer coatings. Such an influence may, in turn, adversely affect the biological properties of the scaffolds.16 To avoid these potential disadvantages caused by continuous polymer coatings, researchers have added bioactive nanoparticles to the polymer coatings to develop nanocomposite coatings that can toughen inorganic scaffolds without reducing the apatiteforming ability.23,24 For example, a polycaprolactone (PCL) coating containing hydroxyapatite particles24 or BG particles25 has been used to coat biphasic calcium phosphate (BCP) scaffolds and the coated scaffolds exhibited improved mechanical properties remaining highly bioactive. Bioactive glass nanoparticles (BGN) are suitable bioactive fillers for nanocomposites, considering their high bioactivity and large specific surface area.26,27 Compared with their micrometer-sized counterparts, carefully prepared BGN are usually more dispersed and uniform in shape.26 Therefore, highly dispersed and uniform BGN fillers should be beneficial for enhancing the properties of polymer matrices. On the basis of the above rationale, we hypothesized that 45S5 BG scaffolds, a most widely investigated BG-based biomaterial for bone-related applications,7,9 would also achieve enhanced mechanical properties without losing their rapid apatite-forming ability via a BGN-containing nanocomposite coating. A series of spherical copper-containing bioactive glass nanoparticles (CuBGN) have been synthesized in our previous study.28 In this
2. MATERIALS AND METHODS 2.1. Fabrication of Cu-BGN/Gelatin-Coated 45S5 BG Scaffold. 45S5 BG scaffolds (BGS) were fabricated using the foam replica method11 while Cu-BGN were synthesized via a modified Stöber method as reported in our previous study.28 The details of these two methods are provided in the Supporting Information. The composition of Cu-BGN has been determined to be ∼95SiO2-2.5CaO-2.5CuO, in mol %, by inductively coupled plasma atomic emission spectroscopy (ICP-AES).28 Morphology of Cu-BGN was observed using field emission scanning electron microscopy (FE-SEM; Auriga, Carl Zeiss, Germany), while the size of Cu-BGN was determined using ImageJ (NIH, USA) by counting the particles in SEM images (more than 400 particles were counted). The polydispersity index (PDI) of Cu-BGN was characterized by a Zetasizer Nano ZS (Malvern Instruments, UK) instrument operating with a 4 mW HeNe laser (633 nm) and a light scattering detector positioned at 90◦. The sample was measured in deionized water at a concentration of 100 μg/mL. BGS were coated with Cu-BGN/gelatin nanocomposites using dip coating, as described in previous reports with slight modification.17,22 Briefly, gelatin (type A, Sigma-Aldrich, USA) solution (5% w/v) was prepared by dissolving gelatin in deionized water at 50 °C. Cu-BGN were then added to the solution at different ratios (in wt %) of Cu-BGN/ gelatin (5 and 20 wt %) under ultrasonic dispersion. The ratios (in wt %) of Cu-BGN/gelatin were determined based on the ratios used for producing gelatin-based nanocomposites reported in the literature.31 In order to cross-link gelatin, genipin (1 wt %/v) (Wako, Osaka, Japan) was dissolved in ethanol and then added to the Cu-BGN/gelatin suspension (genipin/gelatin, 1 wt %) under continuous stirring at 50 °C.17 Genipin is an effective and widely used cross-linker to improve the mechanical properties and stability of gelatin, while it has been reported to be toxic to cells at a relatively high concentration.17 BG scaffolds were then immersed in the suspension and hold for 2 min. The coated scaffolds were then dried at room temperature for 3 days. The obtained scaffolds were designated as 5Cu-BGS and 20Cu-BGS according to the ratios (in wt %) of Cu-BGN/gelatin used (5 and 20 wt %). Pure-gelatincoated BG scaffolds were designated as 0Cu-BGS, which were fabricated as described above without the addition of Cu-BGN. 2.2. Characterization. 2.2.1. Microstructure and Porosity. The microstructure of the scaffolds was characterized using FE-SEM. The samples were observed without being sputter-coated. Energy-dispersive X-ray spectroscopy (EDS) was also performed during the SEM observation. The porosity of the scaffolds was determined by using a liquid displacement method.32 Ethanol was used as the displacement liquid, considering its insignificant influence on the scaffolds. Briefly, a dried sample was soaked in a cylinder containing ethanol (volume V1) prior to the treatment of vacuum in order to replace all the air in the pores with 1547
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2.5. Cell Culture. Mouse preosteoblastic cell lines (MC3T3-E1) were cultured in alpha Minimum Essential Medium (α-MEM; Gibco, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS), 1% penicillin (100 U.mL−1) and streptomycin sulfate (100 mg.mL−1) (Gibco BRL, USA). The cells were cultured in a humidified atmosphere at 37 °C and 5% CO2. When the cells were grown to confluence in 75 cm2 culture flasks (Nunc, Denmark), they were harvested with Trypsin/EDTA (Gibco, Germany) and counted using a hemocytometer (Roth, Germany). The culture medium was replaced every 3 days. 2.6. In Vitro Cytotoxicity, Cell Proliferation, and Attachment. All scaffolds were sterilized using ethylene oxide before being stored in sterilized sealed centrifuge tubes for further cell biology experiments. The cytotoxicity of the scaffolds (6 mm × 6 mm × 4 mm) toward MC3T3-E1 cells was evaluated using the MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) assay. In brief, the scaffolds were soaked in α-MEM to regulate the pH value to stability prior to the seeding of cells. Each scaffold in a 24-well plate was seeded with 100 μL of cell suspension (1 × 106 cells per mL) and then placed in an incubator at 37 °C and 5% CO2 for 4 h to allow cell adhesion. Subsequently, 900 μL of α-MEM supplemented with 10% FBS was added to the well plate for further culture. After 1 day and 5 days of culture, 10 μL of the MTT solution (5 mg.mL−1) was added and the samples were incubated at 37 °C for further 4 h to form MTT formazan. The supernatant was then removed and 150 μL dimethyl sulfoxide (DMSO, Sinopharm, Shanghai, China) was added per well to dissolve the formed formazan. The supernatant was then transferred into 96-well plates for the measurement of the optical density (OD) at 492 nm with a spectrophotometer (Synergy HT Multidetection Microplate, USA). To evaluate cell attachment on the scaffolds, we seeded 100 μL of cell suspension (1 × 106 cells per mL) on each scaffold in a 24-well plate and the cells were allowed to adhesion for 4 h. Subsequently, 900 μL of αMEM supplemented with 10% FBS was added. After 24 h of incubation, the samples were washed gently three times with PBS to remove the unattached cells. The cells still remaining on the scaffolds were then fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells attached on the scaffolds were stained with fluorescein isothiocyanate (FITC)Phalloidin (Sigma-Aldrich, St. Louis, USA) for 60 min for detecting the cytoskeleton and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride) (Sigma-Aldrich) with 5 min for observing the nuclei. Morphological characteristics of cells were visualized using a confocal laser scanning microscopy (Leica Microsystems TCS SP2, Germany). The proliferative effect of the scaffolds on MC3T3-E1 cells was determined by using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan). The seeding procedure for the CCK-8 assay was similar to that of the MTT assay, but the density of seeded cells was 5 × 104 cells per well. Briefly, after culture with cells for predetermined times (1 day, 7 days, and 14 days), the scaffolds were transferred to a new 24-well plate. CCK-8 solution (10 μL per well) was then added prior to further culture of 4 h. 100 μL of supernatant was then collected and read by using a microplate reader (Synergy HT, USA) at 450 nm to obtain the ODs. The cell proliferation was expressed as relative proliferation rates, in which the calculated ODs at days 7 and 14 were normalized to those at day 1 for each group.34 2.7. ALP Activity. The scaffolds in a 24-well plate were seeded with 100 μL of cell suspension (1 × 106 cells per mL). After incubation for 4 h at 37 °C, 900 μL of α-MEM, supplemented with vitamin C (50 μg/mL), 0.01 M β-glycerophosphate, and 100 nM dexamethasone, was added to each well. The scaffolds were further cultured with MC3T3-E1 cells for 3 and 14 days. The medium was exchanged every 2 days. The ALP activity was determined using an ALP Kit (Nanjing Jiancheng, China) according to the manufacturer’s instruction. Briefly, at each predetermined time point, the scaffolds were rinsed with PBS solution and lysed with 0.1% Trion X-100 solution for 30 min at 4 °C. 50 μL of supernatant was then mixed with 50 μL chromogenic substrate and cultured for 30 min at 37 °C. The reaction was stopped by adding 100 μL of terminal liquid according to the manufacturer’s protocol. ALP activity was measured spectrophotometrically with a microplate reader at an absorbance wavelength of 520 nm.
ethanol. The total volume (V2) of the ethanol and scaffold was recorded when no bubbles emerged. Subsequently, the scaffold was removed from the cylinder and the volume of residual liquid was recorded as V3. The porosity (open pores) ε was determined as
ε = (V 1 − V 3)/(V 2 − V 3)100%
(1)
The measurement was carried out in triplicate to determine the porosity. 2.2.2. Structural Characterization. Fourier transform infrared spectroscopy (FTIR) spectra were recorded in transmission mode using a Nicolet 6700 FTIR spectrophotometer (Thermo Scientific, USA) under ambient conditions. The spectra were collected between 2400 and 400 cm−1 at a resolution of 4 cm−1. For the FTIR measurements, the scaffolds were ground, mixed with KBr (spectroscopy grade, Merck, Germany) at a weight ratio of 1:100, and pressed into pellets. X-ray diffraction (XRD) was performed using a D8 ADVANCE X-ray diffractometer (Bruker, USA) in a 2θ range of 20−80° with Cu Kα radiation. A step size of 0.014° with a dwell time of 1 s per step was used. For the XRD measurements, the scaffolds were ground and measured in powder form. 2.2.3. Thermal Degradation. The thermal degradation of the scaffolds was measured using thermogravimetric analysis (TGA) by a thermogravimetric analyzer Q5000IR (TA Instruments, USA). The measurement was conducted from room temperature to 800 °C with a heating rate of 10 °C/min under an inert nitrogen atmosphere. 2.2.4. Compressive Strength. The compressive strength of the scaffolds (10 mm × 8 mm × 8 mm) was determined using a Zwick/Roell Z050 mechanical tester. The applied crosshead speed was 0.5 mm/min and a load cell of 1 kN was used. The compressive tests were conducted until the strain reached 70%. The maximum stress in the stress−strain curve before densification was measured. The tests were performed on at least five samples, and the results were shown as a mean value ± standard deviation (S.D.). 2.3. In Vitro Apatite Formation. The in vitro apatite formation of the scaffolds during the exposure to simulated body fluid (SBF) was assessed according to the protocol proposed by Kokubo et al.33 Briefly, the scaffolds were immersed in SBF at a ratio of 1 mg/mL at 37 °C and 90 rpm for predetermined times. SBF was replaced twice per week during the measurement. At each predetermined time point, the scaffolds were removed from SBF and rinsed with deionized water gently before drying at room temperature. The apatite formation on the scaffolds was assessed by FE-SEM, FTIR, XRD, and EDS. The sample preparation for these measurements was the same as described above. 2.4. In Vitro Degradation. The weight loss of the scaffolds was performed in SBF to evaluate the in vitro degradation. Briefly, the scaffolds (10 mm × 8 mm × 8 mm) were soaked in 50 mL of SBF at 37 °C and 90 rpm for different times. At each predetermined time point, the scaffolds were carefully withdrawn from the solution and rinsed with ethanol prior to drying at room temperature. The weight loss was calculated using eq 2:
weight loss (%) = (M1 − M 2)/M1·100%
(2)
where M1 and M2 are the mass of the scaffolds before and after soaking in SBF, respectively. The changes of pH value of the SBF solution containing the scaffolds at the early stage of immersion (within 3 days) were also measured. During the pH measurement, the SBF solution was not changed. The ion release of the scaffolds in phosphate-buffered saline (PBS; pH 7.4) was also measured to evaluate the degradation behavior of the coated scaffolds. 20Cu-BGS was selected as the representative sample for ion release test considering its higher amount of Cu-BGN, in which the concentrations of Si, Ca and Cu ions were measured. Briefly, 70 mg of the samples were soaked in 10 mL of PBS. At every predetermined time point, 5 mL of the supernatant was collected and the samples were replenished to 10 mL with fresh PBS. The ion concentration was then determined by ICP-AES (IRIS 1000, Thermo Elemental, USA). To evaluate the leaching of Cu-BGN from the composite coating, we checked the supernatant at selected time points (8 h and 14 days) with FE-SEM. 1548
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Figure 1. SEM images at different magnifications and EDS spectra of BG scaffolds before and after the coating (presence or absence of Cu-BGN). (a) Uncoated 45S5 BG scaffold; (b) pure-gelatin-coated scaffold (0Cu-BGS); Cu-BGN/gelatin nanocomposite-coated scaffolds (c) 5Cu-BGS and (d) 20Cu-BGS with an SEM image of Cu-BGN inseted.
the scaffolds prepared using the form replica method.11,17,19 After coating with gelatin, the scaffolds still retained their highly porous structure (Figure 1b), though their porosity was slightly reduced from 96 to 92%. With the incorporation of Cu-BGN in the gelatin coating regardless of the concentration of particles, the pore structure of the scaffolds was not significantly affected (Figure 1c, d). The coated scaffolds remained highly interconnected and their macroporous structure was maintained. Only a limited number of pores were clogged by the coating. The porosity of 5Cu-BGS and 20Cu-BGS was the same and it was determined to be 91%, which was not significantly different to that of 0Cu-BGS. Thus, the porosity of the coated scaffolds was
2.8. Statistical Analysis. All quantitative data were expressed as the mean ± standard deviation. Statistical analysis was performed with oneway analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
3. RESULTS AND DISCUSSION 3.1. Cu-BGN Can Be Incorporated into Gelatin Coating without Affecting the Porous Structure of 45S5 BG Scaffold. Figure 1 shows representative SEM images of BG scaffolds before and after coating. The uncoated BG scaffolds exhibited a highly interconnected pore structure with a pore size of 200−600 μm (Figure 1a), which is the typical morphology of 1549
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Figure 2. Changes of structural characteristics in the 45S5 BG scaffolds after incorporating Cu-BGN in the coating (a) TGA curves, (b) FTIR spectra, and (c) XRD patterns.
scaffolds occurred between 60 and 120 °C, which is likely due to the removal of residual water in the coating. The following weight loss starting at approximately 250 °C could be attributed to the thermal degradation of gelatin.36 The final weight loss at 800 °C of 0Cu-BGS, 5Cu-BGS, and 20Cu-BGS were 24, 20, and 15%, respectively. 45S5 BG scaffold and Cu-BGN could not be thermally decomposed in the tested temperature range (25 to 800 °C), therefore, the main weight loss could be ascribed to the removal of gelatin. Thus, the difference in weight loss of the coated scaffolds indicated the presence of thermally stable CuBGN in the coatings. The real amount of Cu-BGN in the scaffolds can thus be determined by comparing the weight loss of the scaffolds with or without Cu-BGN incorporation in the TGA measurements. However, it is challenging to determine the real ratios (in wt %) of Cu-BGN/gelatin in the coated scaffolds, because gelatin usually has residual products (e.g., carbon residual) after the TGA measurement (heating up to 800 °C).37 The presence of gelatin residual products prevents the accurate determination of the weight ratio of gelatin in the scaffolds, but relative ratios (in wt %) of Cu-BGN/gelatin, that were affected by the presence of residual products related to the weight loss over 800 °C, could be determined. The relative ratios (in wt %) of CuBGN/gelatin could then be determined as ∼11% and ∼37% in 5Cu-BGS and 20Cu-BGS, respectively, according to the method described previously.37 Although the relative ratios (in wt %) are higher than the designed ratios (5% and 20%), the real ratios should be closer to the designed ones considering the presence of an uncounted amount of gelatin residuals. Figure 2b shows the FTIR spectra of the scaffolds before and after coating. All scaffolds exhibited typical bands of crystallized 45S5 BG. Two main vibrational bonds of Si−O−Si (460 and 1000−1200 cm−1) could be observed while three strong peaks
retained at a level that is beneficial for cellular and vascular infiltration as well as for tissue ingrowth.35 Unlike polymer solution with high viscosity, the relatively low viscosity of gelatin solution facilitated the solution flowing inside the porous structure of the scaffolds, which was beneficial for reaching a homogeneous continuous coating and for reducing the possibility of blocking the pores. Spherical particles were observed on 5Cu-BGS and 20Cu-BGS (Figure 1), whereas no particles were seen on noncoated scaffolds and 0Cu-BGS. These particles could thus be regarded as Cu-BGN. As expected, a larger number of particles were seen on 20Cu-BGS than 5Cu-BGS. Notably, these Cu-BGN were homogeneously dispersed in the coating, which could be due to the high dispersity and stability of Cu-BGN in aqueous gelatin solution inducing a homogeneous suspension during the coating process. As shown in Figure S1, Cu-BGN were homogeneous in size (∼430 nm) and shape. Their PDI in water was measured to be 0.103, which indicates the high dispersity of Cu-BGN. Such a homogeneous morphology and high dispersity of Cu-BGN were beneficial for the formation of a homogeneous suspension for preparing the coatings. After the incorporation of Cu-BGN in the gelatin coating, the existence of Cu was confirmed in the EDS spectra of 5Cu-BGS and 20Cu-BGS (Figure 1). The observation of C in the EDS spectra of 0Cu-BGS, 5Cu-BGN, and 20Cu-BGS indicated the presence of gelatin coating. In addition to the direct SEM observation and EDS spectra, the successful incorporation of Cu-BGN into the gelatin coating was also proven by other characterization methods. Figure 2a shows the TGA results of the uncoated and coated scaffolds. Two obvious weight loss curves were observed in the coated scaffolds, whereas no significant change in weight for the uncoated one was observed. The first weight loss of the coated 1550
DOI: 10.1021/acsbiomaterials.8b00051 ACS Biomater. Sci. Eng. 2018, 4, 1546−1557
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ACS Biomaterials Science & Engineering located at around 526, 574, and 620 cm−1 could be attributed to P−O bending vibration bonds in crystalline phosphate.38,39 In comparison to the spectra of uncoated scaffolds, three new bands at 1440, 1540, and 1660 cm−1 were observed in the spectra of 0Cu-BGS, 5Cu-BGN and 20Cu-BGS, which could be assigned to the gelatin amino acids C−H bond, amide N−H bending vibration bond (amide II), and amide CO stretching vibration bond (amide I), respectively.17,40 The observation of these bands indicated the presence of genipin cross-linked gelatin in the coated scaffolds. No significant difference in the FTIR spectra could be observed among 0Cu-BGS, 5Cu-BGS, and 20Cu-BGS, as the characteristic bands of Cu-BGN in FTIR spectra were likely overlapped by the characteristic bands of 45S5 BG.28,38 Similarly, no significant difference could be observed in the XRD patterns of the uncoated and coated scaffolds (Figure 2c). Only the characteristic peaks related to the crystalline phases Na4Ca4(Si6O18) and Na4Ca4(PO4)2SiO4 which usually form in sintered 45S5 BG could be seen.11 Because of the amorphous nature of Cu-BGN that has been proven by the XRD result in our previous study,28 no crystalline phases related to the nanoparticles were observed in the XRD patterns. 3.2. Presence of Cu-BGN Does Not Affect the Mechanical Improvement Caused by Gelatin Coating. Figure 3a shows the compressive strength of scaffolds before and after coating. Uncoated scaffolds exhibited a compressive
strength of 0.03 ± 0.01 MPa, whereas 0Cu-BGS, 5Cu-BGS, and 20Cu-BGS reached 1.3 ± 0.2, 1.2 ± 0.2, and 0.7 ± 0.3 MPa, respectively. As expected, the gelatin-based coating significantly enhanced the compressive strength of BG scaffolds.17,41 Notably, the enhancement effect was not significantly different between 0Cu-BGS and 5Cu-BGS, which indicated that the incorporation of Cu-BGN in the gelatin coating at a proper concentration would not affect the mechanical improvement effects caused by the gelatin coating. However, the presence of Cu-BGN in the gelatin coating at a relatively high concentration (20Cu-BGS) seems to compromise the mechanical improvement. As discussed above, the coated scaffolds still exhibited a high porosity of ∼91% that was similar to that of human cancellous bone (∼90%), but their compressive strength (∼1.2 and ∼0.7 MPa) was higher than the lower bound value of compressive strength of human cancellous bone (∼0.15 MPa).42 Notably, all coated scaffolds remained an integrated structure staying in one piece after compression tests, whereas uncoated scaffolds were fully crushed after the test (Figure 3b), which indicated the nanocomposite coating could also improve the toughness of the scaffolds. It is known that the lack of effective sintering and densification is one of the main challenges inhibiting the clinical applications of highly porous 45S5 BG scaffolds, as the narrow sintering window of 45S5 BG causes the occurrence of crystallization prior to the full densification of the glass.11,43 The existence of voids between the grains and the formation of microcracks significantly reduce the mechanical strength. Additionally, the form replica method itself leads to voids in struts after calcination, which can also reduce the mechanical strength of the scaffolds.11 Natural polymers have been widely used as coatings to increase the mechanical properties of bioceramic/bioactive glass scaffolds.16 By filling the microcracks and hollow voids in the struts, polymers can densify the constructs and may also toughen the scaffolds exploiting the intrinsic toughness of the polymers.16,44,45 This improvement in mechanical properties has been well explained by the micrometer-scale crack-bridging mechanism.44 Inorganic/organic nanocomposite coatings can also achieve a desired mechanical improvement, in which inorganic fillers may also bring additional novel functionalities.24,25 However, the concentration of inorganic fillers in the polymer coating should be carefully controlled in order to achieve effective mechanical improvement. At relatively low concentrations, the inorganic fillers do not impede the polymer phases from infiltrating (healing) the microcracks and voids of the scaffold struts, which is consistent with the results of compressive strength of 5Cu-BGS (Figure 3). However, with increasing the concentration of Cu-BGN in the gelatin coating, those fillers might interfere with the flow of gelatin solution into the microcracks and voids of the struts, which, in turn, reduces the effectiveness of the coating to improve the mechanical properties.25 Although the mechanical properties of 45S5 BG scaffolds have been significantly improved after the coating of the nanocomposite, it is still challenging to use the scaffolds for load-bearing bone applications, given that the compressive strength is still lower than the required strength for load-bearing bone repair/regeneration (e.g., the compressive strength of human cortical bone: 100−150 MPa).46 The current scaffolds are thus more suitable for applications in nonloadbearing sites. Further optimization and modification of the coating, such as tuning the thickness of the coating, will be carried out in future studies to enhance the mechanical properties. 3.3. Incorporation of Cu-BGN Facilitates Apatite Formation on Gelatin Coating. Figure 4 shows SEM images
Figure 3. (a) Compressive strength of BG scaffolds before and after nanocomposite coating; (b) digital photographs of uncoated and coated scaffolds before (inserted) and after compressive strength test showing different mechanical failure behaviors. 1551
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ACS Biomaterials Science & Engineering
Figure 4. SEM images at different magnification and EDS spectra of the coated 45S5 BG scaffolds after immersion in SBF with inseted highmagnification images. (a) 0Cu-BGS in SBF for 1 day, (b) 5Cu-BGS in SBF for 1 day, (c) 20Cu-BGS in SBF for 1 day, (d) 0Cu-BGS in SBF for 3days, (e) 5Cu-BGS in SBF for 3 days, (f) 20Cu-BGS in SBF for 3 days.
of the coated scaffolds after immersion in SBF for 1 day and 3 days. The deposition could be seen on 5Cu-BGS and 20Cu-BGS after their immersion in SBF for 1 day, while no obvious deposition was found on 0Cu-BGS. After 3 days of immersion, the deposition could be seen on 0Cu-BGS while all the visible areas of the images of 5Cu-BGS and 20Cu-BGS were covered. Additionally, this deposition exhibited a cauliflower-like shape, which is a typical morphology of HA forming on BG after exposure to SBF.38 Phosphorus appeared in the EDS spectra of Cu-BGN/gelatin coated scaffolds after immersion in SBF. The Ca/P ratio increased with immersion time in SBF and this ratio reached a value of ∼1.67 in 5Cu-BGS and 20Cu-BGS after 3 days of immersion in SBF, which is close to the stoichiometric Ca/P ratio of HA. Notably, sodium and magnesium were also found in the EDS spectra, which were originally from SBF and then incorporated on the scaffolds. In addition, calcium in the formed apatite crystals could be partially substituted by magnesium from SBF.40 FTIR spectra (Figure 5) also confirmed the formation of HA in the scaffolds after immersion in SBF, as two characteristic bands at 596 and 604 cm−1 corresponding to P−O bonds in HA were observed in the spectra of 5Cu-BGS and 20Cu-BGS after 1 day of immersion in SBF. However, these bands only appeared in 0Cu-BGS after 3 days of immersion in SBF. Moreover, the presence of a band at 876 cm−1 and dual broad bands between
Figure 5. FTIR spectra of different scaffolds after immersion in SBF for different days indicating the formation of HCA and the influence induced by the presence of Cu-BGN.
1400 and 1455 cm−1 being attributed to the stretching vibration of C−O bond, suggesting that the formed apatite was carbonated hydroxyapatite (HCA) rather than stoichiometric HA.17 These 1552
DOI: 10.1021/acsbiomaterials.8b00051 ACS Biomater. Sci. Eng. 2018, 4, 1546−1557
Article
ACS Biomaterials Science & Engineering
Figure 6. (a) Weight loss in SBF of 45S5 BG scaffolds before and after the coating; (b) representative ion release behavior in PBS of Cu-BGN/gelatin nanocomposite coated scaffolds (20Cu-BGS); (c) pH values of SBF solution after the immersion of the scaffolds.
Acceleration of apatite formation on polymer coating due to the incorporation of Cu-BGN has also been observed in polycaprolactone coated magnesium implants.29 Although the apatite formation was promoted by the addition of Cu-BGN in the gelatin coating, such incorporation had no significant effect on the degradation of the coated scaffolds. Figure 6a shows the weight loss of the scaffolds in SBF. The weight of all scaffolds decreased over time in SBF. After immersion for 21 days, the weight loss of uncoated scaffolds, 0Cu-BGS, 5Cu-BGS, and 20Cu-BGS reached ∼38, 47, 48, and 53%, respectively, indicating that all scaffolds were degradable in SBF. The coated scaffolds exhibited greater weight loss than the uncoated scaffold, which could be due to the rapid dissolution of gelatin. Although the cross-linking of gelatin improved its resistance to the aqueous environment, the cross-linked gelatin still suffered from relatively rapid dissolution/degradation.17 It has been reported that the weight loss of genipin cross-linked gelatin could reach 62% after 7 days in SBF, which was significantly greater than the weight loss of 45S5 BG scaffolds in SBF.17 These facts could account for the greater weight loss of the coated scaffolds than the uncoated scaffolds in this study. However, the addition of Cu-BGN in the coating had no significant effects on the weight loss of the scaffolds, which was understandable considering the small amount of Cu-BGN in the coating. Cu-BGN are also degradable in aqueous solution. It has been well-documented that the degradation of 45S5 BG scaffolds can cause the rise of the pH of biological fluids.49 In this study, the presence of gelatin coating reduced the extent of pH rise of SBF at the early stage of immersion (
