Incorporation of Cu-Containing Bioactive Glass Nanoparticles in

Subscriber access provided by UNIV OF DURHAM

Characterization, Synthesis, and Modifications

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., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00051 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Incorporation of Cu-containing bioactive glass nanoparticles in gelatin coated scaffolds enhances bioactivity and osteogenic activity

Kai Zhenga*, Jingjing Wub*, Wei Lic, Dirk Dippoldd, Ying Wanb#, Aldo R. Boccaccinia#

a

Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen, Germany

b

College of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, P. R. China

c

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI00014 Helsinki, Finland d

Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Martensstrasse 7, 91058 Erlangen, Germany

*These two authors contributed equally to this paper #

Corresponding authors:

[email protected] (A. B.); Tel: +49 9131 85 28601; Fax: +49 9131 85 28602 [email protected] (Y. W.); Tel.: + 86 27 87792147; Fax: +86 27 87792234

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: 95SiO2-2.5CaO-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 one day of immersion in simulated body fluid. The osteogenic activity as indicated by the ALP

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

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 CuBGN 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 CuBGN/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 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 supporting and guiding tissue ingrowth 4. These interactions between cells and scaffolds are regulated by many factors, such as the composition property and surface topography of the scaffolds 4,5. Among numerous candidate materials, bioactive glasses (BG) 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 BG 8. 45S5 BG (45SiO2+24.5Na2O+24.5CaO+6P2O5, in wt.%), the first developed BG, can bond to bone and then induce bone growth 9. 45S5 BG is also reported to be able to facilitate vascularization and to be antibacterial at proper concentrations


10

. 45S5 BG can be

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 due to their high porosity and poor sintering behavior of 45S5 BG 11. Towards 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 pre-designed 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 structure16. 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 synthetic 19 and natural ones 17 or their combination 20 have been widely used to coat 45S5 BG scaffolds. These polymers can be coated on 45S5 BG scaffolds in the form of films/membranes

20

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 also be influenced by polymer coatings. Such an influence may, in turn, adversely affect the biological properties of the scaffolds 16. In order to avoid these potential disadvantages caused by continuous polymer coatings, bioactive nanoparticles have been added to the polymer coatings to develop nanocomposite coatings that can toughen inorganic scaffolds without reducing the apatite-forming ability 23,24

. For example, a polycaprolactone (PCL) coating containing hydroxyapatite particles 24 or

BG particles

25

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 bioactive and large specific surface area


26,27

. Compared with their


Page 4 of 31

micron-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. Based on 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 BGNcontaining nanocomposite coating. A series of spherical copper-containing bioactive glass nanoparticles (Cu-BGN) have been synthesized in our previous study 28. In this research, we selected the particles with a composition of 95SiO2-2.5CaO-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 enhanced the apatite-forming ability that was comparable with that of the un-coated 45S5 BG scaffold. Additionally, it was investigated if the presence of Cu-BGN at proper concentrations could enhance the osteogenic activity of scaffolds. 2. Materials and methods 2.1. Fabrication of Cu-BGN/gelatin coated 45S5 BG scaffold 45S5 BG scaffolds (BGS: 45SiO2+24.5Na2O+24.5CaO+6P2O5, in wt.%) were fabricated using a foam replica method

11

while Cu-BGN (95SiO2-2.5CaO-2.5CuO, in mol.%) 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) in our previous study

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)


Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 the 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 distilled water at 50 °C. Cu-BGN were then added to the solution at different ratios (in wt.%) of Cu-BGN/gelatin (5 wt.% 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 in a previous study 31. In order to crosslink 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 wt.% and 20 wt.%). Pure gelatin coated 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 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) ɛ was determined as: ɛ = (1 − 3)/(2 − 3) × 100% (1)



Page 6 of 31

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 cm-1 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 K α 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 x 8mm x 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 used to the determination of compressive strength. 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

o

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.


Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 x 8mm x 8 mm) were soaked in 50 mL of SBF at 37oC 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 Eqn (2): Weight loss (%) = (M1 − M2)/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 inductively coupled plasma-atomic emission spectroscopy (ICP-AES; IRIS 1000, Thermo Elemental, USA). In order to evaluate the leaching of Cu-BGN from the composite coating, the supernatant at selected time points (8 h and 14 d) were checked with FE-SEM. 2.5. Cell culture Mouse pre-osteoblastic 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 oC 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 three 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



X 6mm X 4 mm) towards MC3T3-E1 cells was evaluated using the MTT (3-(4,5dimethylthiazol-2-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 (1x106 cells per mL) and then placed in an incubator at 37 oC 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 oC 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 Multi-detection Microplate, USA). To evaluate cell attachment on the scaffolds, 100 μL of cell suspension (1x106 cells per mL) was seeded on each scaffold in a 24-well plate and the cells were allowed to adhesion for 4 h. Subsequently, 900 μL of α-MEM 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',6Diamidino-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 x 104 cells per well. Briefly, after culture with cells for pre-determined 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 hours. 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 day 7 and 14 were normalized to those at day 1 for each group 34. 2.7. ALP activity


Page 8 of 31

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The scaffolds in a 24-well plate were seeded with 100 μL of cell suspension (1 x 105 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 two 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 oC. 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. 2.8. Statistical analysis All quantitative data were expressed as the mean ± standard deviation. Statistical analysis was performed with one-way 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 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 determined to be 91%, which was not significantly different to that of 0Cu-BGS. Thus the porosity of the coated scaffolds was 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 homogenous continuous coating and for reducing the possibility of blocking the pores. Spherical particles were observed on 5Cu-BGS and 20Cu-BGS (Figure 1) while no particles were seen on non-coated 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 homogenous 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, while no significant change in weight for the uncoated one was observed. The first weight loss of the coated scaffolds occurred between 60 and 120 oC, which is likely due to the removal of residual water in the coating. The following weight loss starting at approximately 250 oC could be attributed to the thermal degradation of gelatin 36. The final weight loss at 800 oC 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 oC), therefore, the main weight loss can be ascribed to the removal of gelatin. Thus, the difference in weight loss of the coated scaffolds indicated the presence of thermally stable Cu-BGN 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 oC) 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


Page 10 of 31

Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 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



Page 12 of 31

gelatin coated scaffold (0Cu-BGS); Cu-BGN/gelatin nanocomposite coated scaffolds (c) 5CuBGS and (d) 20Cu-BGS with an SEM image of Cu-BGN inserted.

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 cm-1 and 1000-1200 cm-1) could be observed while three strong peaks 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 C=O 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 0CuBGS, 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

. Due to 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.


Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. The 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.

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 while 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 5CuBGS, which indicated that the incorporation of Cu-BGN in the gelatin coating at 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 MPa and ~0.7 MPa) was higher than the lower bound value of compressive strength of human cancellous bone



Page 14 of 31

(~0.15 MPa) 42. Notably, all coated scaffolds remained an integrated structure staying in one piece after compression tests, whereas un-coated 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 micron-scale crackbridging 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 concentration, 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 nonload-bearing 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.


Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

3.3. Incorporation of Cu-BGN facilitates apatite formation on gelatin coating Figure 4 shows SEM images 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



Page 16 of 31

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 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 results clearly

indicate that the presence of Cu-BGN in the gelatin coating induced (accelerated) the apatite formation on the surface of the scaffolds. 5C

Figure 4. SEM images at different magnification and EDS spectra of the coated 45S5 BG scaffolds after immersion in SBF with inserted high magnification images. (a):0Cu-BGS in SBF


Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

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.

Although polymer coatings can significantly improve the mechanical properties of BG-based scaffolds 16, their presence may delay the apatite formation 17,20, as the polymer coatings act as barriers preventing the effective interaction between body fluids and the BG surface. It is known that 45S5 BG scaffolds themselves are highly bioactive and HCA can form on the scaffolds within 1 day of immersion in SBF 17. However, after the coating with gelatin, the formation of HCA was delayed and this formation was only observed in scaffolds after 3 day immersion (Figure 4). Many efforts have been made to achieve improved mechanical properties without the cost of bioactivity reduction, such as surface functionalization on polymer coatings 47. Development of nanocomposite coatings composed of polymer phases and bioactive fillers is an alternative strategy to address this issue

24,25,48

. In this study, the

incorporation of Cu-BGN in the gelatin coating significantly accelerated the HCA formation that was observed on 5Cu-BGS and 20Cu-BGS after immersion in SBF for 1 day. Given the rapid formation of apatite on 5Cu-BGS and 20Cu-BGN, the bioactivity of the Cu-BGN/gelatin nanocomposite coated BG scaffolds is considered to be comparable with uncoated 45S5 BG on which HCA also forms within 1 day of immersion in SBF

11,17

. The acceleration of HCA

formation is due to the incorporation of Cu-BGN that have been proven to be highly bioactive in our previous study 28, in which cauliflower-like HCA crystals could fully cover the



surface of Cu-BGN pellets within 3 days of immersion in SBF. In 5Cu-BGS and 20Cu-BGS, CuBGN were available on the surface of the coating (Figure 1) and thus the ion exchange between Cu-BGN and SBF towards apatite formation could take place earlier. Acceleration of apatite formation on polymer coating due to the incorporation of Cu-BGN has also been observed in polycaprolactone coated magnesium implants 29.

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.

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 un-coated scaffold, which could be due to the rapid dissolution of


Page 18 of 31

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gelatin. Although the crosslinking of gelatin improved its resistance to the aqueous environment,

the

crosslinked

gelatin

still

suffered

from

relatively

rapid

17

dissolution/degradation . 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 CuBGN 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 (

Incorporation of Cu-Containing Bioactive Glass Nanoparticles in

Recommend Documents