Investigation of in vitro bioactivity and antibacterial activity of manganese-doped spray pyrolyzed bioactive glasses

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Abstract

Bioactive glasses (BGs) are of great interest in fields of medical engineering owing to their biocompatibility, biodegradability and bioactivity. In addition, spray pyrolysis was demonstrated as a novel way to obtain BG particles with various compositions and dopants while controlling the particle morphologies. In the present study, BGs doped with Manganese (Mn), an ion that has been shown to be osteoconductive, were prepared by spray pyrolysis method. Characterizations of crystallographic structure, particle morphology and chemical compositions were carried out by X-ray diffractometer, scanning electron microscope, transmission electron microscope and energy dispersive spectroscopy, respectively. Furthermore, evaluation of in vitro bioactivity was performed regarding the formation of hydroxyapatite layer while the colony count method was used to examine the antibacterial activity against E. coli bacteria. Finally, the results show that both bioactivity and antibacterial activity were enhanced with dopant of Mn and related mechanisms were discussed.

Introduction

Since Hench et al. introduced bioactive glass (BG) in 1969 [1], its bioactive properties have been investigated in various studies. Notably, the hydroxyapatite layers that are formed on its surface when it is implanted into the human body chemically bond to human bones, thus reducing rejection and inflammation [2], [3]. Furthermore, owing to their superior properties such as biocompatibility, biodegradability, and bioactivity, BGs have attracted considerable attention and have been used in various biomedical applications such as tooth fillers [4], bone implants [5], and drug carriers [6]. However, during the stages of bone reconstruction, the development of bacterial infection is a major complication. As bone implants, BGs have a significant disadvantage, which is the lack of antibacterial activity that may lead to severe bacterial infection after surgery [7]. To reduce the risk of complications, BGs with antibacterial activity are considered a promising candidate.

Studies have reported various metal ion dopants that exhibit a broad range of antibacterial activity, such as silver ions, zinc ions, copper ions, manganese ions, etc., which can deactivate the cell membrane of different bacteria, including Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) [8], [9]. Among these ions, silver ions are the most well-known agent with superior thermal stability and non-toxicity at low concentration. Nawaz et al. incorporated Mn in BG, which provided antibacterial effects against Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), and S. aureus [10]. In addition, irrespective of the antibacterial activity, Mn2+ ions are crucial to ensure good metabolism of the bone (bone density, bone formation, and bone resorption) [11]. For instance, Fujitani et al. improved the proliferation and adhesion of osteoblast cells using Mn-containing hydroxyapatite [12]. Furthermore, researchers have reported that prolonged Mn deficiency is associated with osteoporosis [13], and low serum Mn levels have been found in osteoporotic subjects, indicating its significance for the development of the skeletal system [14].

Based on previous studies, several preparation methods have been demonstrated for the synthesis of BGs, such as the conventional glass melting process [1], [2], [3], sol-gel method [15], [16], [17], and spray-assisted techniques [18], [19], [20]. Among these methods, Miola et al. modified conventional melt-derived glass SiO2-P2O5-CaO-MgO-Na2O-K2O with different amounts of MnO and demonstrated its potential in osteoblast proliferation [21]. Moreover, Barrioni et al. reported that sol-gel derived Mn-doped bioactive glasses can enhance the proliferation and viability of osteoblastic cells while confirming their bioactivity [22]. However, for the conventional glass melting process, a high calcination temperature of up to 1500 °C is commonly used, which may cause structural inhomogeneity and difficulty in maintaining high purity [15]. Instead, the sol-gel method offers an alternative, allowing a low calcination temperature of approximately 600 °C with much higher purity control. Still, the disadvantages of long preparation time and batch production in the sol-gel method make mass production for BGs hard to accomplish. To overcome these disadvantages, we aimed at synthesizing Mn-doped BG specimens using spray pyrolysis. The method offers rapid calcination, faster fabrication, and continuous process compared to conventional melt-derived and sol-gel methods [23].

In this study, un-doped and 1, 3, and 5 mol% Mn-doped BG specimens were prepared, and the crystallographic structures, particle morphologies, and chemical compositions were characterized via X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive spectroscopy (EDS), respectively. Additionally, in vitro bioactivity tests were conducted, and both SEM and Fourier transform infrared spectroscopy (FTIR) were used for examination. Further, the antibacterial activity was evaluated against E. coli with statistical measurements of inhibition rate. The present paper describes these methodologies and discusses the formation mechanisms and related properties of BG specimens.

Section snippets

Synthesis

To prepare BG specimens with a composition of 58S (60 mol% SiO2, 36 mol% CaO, and 4 mol% P2O5), at first un-doped precursor solutions were prepared. This involved dissolving 37.49 g tetraethyl orthosilicate (TEOS, Si(OC2H5)4, 99.9 wt%, Showa, Japan), 25.50 g calcium nitrate tetrahydrate (CN, Ca(NO3)2.4H2O, 98.5 wt%, Showa, Japan), and 4.37 g triethyl phosphate (TEP, (C2H5)3PO4, 99.0 wt%, Alfa Aesar, UK) into 60.00 g ethanol as the source of Si, Ca, and P, respectively. For the Mn-doped BG

Results

The crystallographic structure is discussed with the help of Fig. 1, which shows the XRD patterns of un-doped, and 1, 3, and 5 mol% Mn-doped BG specimens prepared by spray pyrolysis. For the un-doped BG specimen, no distinct diffraction peaks were seen in the XRD pattern; instead, a broad band was observed between the angles 20° and 40°. This indicates that the crystallographic structure of the un-doped BG specimen is amorphous. In addition, all Mn-doped BG specimens showed results that are

Discussion

First, the morphologies of the BG specimens are discussed. Previous studies have identified various factors that might influence the particle morphology during a spray pyrolysis process; e.g., the concentration of the solution, the properties of the precursors, or the size of the atomized droplets [27], [28], [29]. In addition, two dominant formation mechanisms are known: “one-particle-per-droplet” and “gas-to-particle” [30]. According to the SEM images in Fig. 2, both un-doped and Mn-doped BG

Conclusions

In this study, successful synthesis of un-doped, and 1, 3, and 5 mol% Mn-doped BG specimens using spray pyrolysis was demonstrated. Results from SEM and TEM measurements lead to conclusions that both un-doped and Mn-doped BG specimens exhibit three particle morphologies, namely solid sphere, porous sphere, and hollow sphere. In addition, bioactivity tests confirmed that all BG specimens were bioactive with the formation of hydroxyapatite when the specimens were immersed in SBF for 24 h. Results

CRediT authorship contribution statement

Chun-Fu Tseng: Data curation, Formal analysis, Visualization. Yu-Chieh Fei: Data curation, Formal analysis, Visualization. Yu-Jen Chou: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors acknowledge the financial support from the Ministry of Science and Technology of Taiwan (Grant number: MOST 108-2218-E-011-035).

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