Nano strontium-loaded bioactive glasses for bone tissue engineering: preparation and bioactivity assessment in simulated body fluid

Bioactive glasses (BGs) have been recently used for bone tissue engineering aims. In this paper, we reported the preparation of nano strontium (NanoSr) loaded BGs. The prepared glasses was characterized and then was tested for bioactivity verification in simulated body fluid (SBF). The scanning microscopic images showed the creation of a hydroxyapatite layer on the sample (BGs and NanoSr loaded BGs) after 2 weeks. However, the hydroxyapatite formation was more for NanoSr loaded BGs. We suggested that the prepared BGs can be used for bone tissue engineering purposes owing to their bioactivity in SBF.

Keywords: Bone formation, Nanoparticles, Bioactive glass, Strontium, Hydroxyapatite

1. Introduction

Bone regeneration and bone tissue engineering have shown an increasingly significant role in the dentistry field. According to reports, the occurrence of bone defects became double by 2020 [1]. There are some traditional ways such as autograft, allograft and xenograft that have been shown limitations including donor site injury, rejection as well as disease spreading [2]. Some alternative ways such as using natural or synthetic biomaterials have been suggested for bone tissue engineering applications [3]. Bioactive glasses (BGs) as a main group of biomaterials have revealed good attention because of their outstanding properties. They are osteoinductive and osteoconductive bioceramics [3, 4]. They have gained numerous uses as a bone block, cement and bone void filler to remove problems such as fractures, bone cysts and tumors [5].

Recently, some inorganic materials in the ionic form are added to the bioactive glasses to progress their bioactivity. Improved biological possessions, such as better angiogenesis and better wound healing can also be achieved by adding inorganic ionic species. Reports have shown the main role of strontium (Sr²⁺) as ionic specie for improving the osteogenic activity of BGs. Strontium Ranelate (SrR), for example, has been applied for years for the treatment of osteoporosis [6, 7]. It has also been revealed in reports that Sr has an important role in increasing bone density and reducing fracture risk mammals [8, 9]. Scientific reports also show Sr ions are able to accelerate bone regeneration via a two-step mechanism; increasing of new bone deposition by osteoblasts and at the same time decreasing bone resorption of bone by osteoclasts [10, 11]. Besides, angiogenesis shows main role in accelerating new bone formation. It is regulated by several growth factors and some conditions such as Hypoxia [11]. Reports have shown for inducing angiogenesis over hypoxia ion releasing glasses can be useful in vitro and in vivo [12, 13]. For example, it has been proposed that Co containing bioactive glasses can simulate a hypoxic condition which is then can activate angiogenesis-related genes and thus help bone tissue regeneration [14, 15].

Nanotechnology in medicine, nanomedicine, offers new ways together outstanding tactics for the detection of oral health-related problems. Nanodentistry is a branch of science that utilizes nano-based approaches to develop new approaches in dental and periodontal areas. It presents the designing of biocompatible dental materials with improved possessions and antimicrobial ability [16-23].

There are a lot of new reports present new data around the effect of Sr in vitro and in vivo. However, physicochemical and biological actions of glasses is still not well identified. So, the chief aim of the current research was to prepare bioactive glasses containing nano Sr for the improvement of its biological actions.

2.1. Glass preparation

Nano Sr was purchased from Nano Kimia Tech company (Tabriz, Iran). All the materials for the preparation of SBF (NaCl, KCl, MgCl₂·6H₂O and CaCl₂) were analytical grade. Acetone was purchased from Merck, Germany.Nano Sr-doped bioactive glasses was synthesized by the sol-gel method according to the method by Imani et al [24]. The combination of powders melted in a platinum-rhodium container for 1 h in an electric furnace (Lenton, UK). After melting, the glasses was quickly put into water to avoid crystallization. When the glasses dehydrated, it was milled according to Kargozr et al  [25].

2.2. Glass characterization

Powder X-ray diffraction (PXRD) test for the samples (BGs and NanoSr loaded BGs) were verified at room temperature by means of an X-ray diffractometer (Siemens, Germany) operating with Cu Kα, a voltage of 50 kV, and a current of  25 mA.

FTIR spectra were determined to approve the chemical structures of samples (BGs and NanoSr loaded BGs) were gained using an FTIR spectrophotometer (Shimadzu 43000, Kyoto, Japan) via the KBr disk technique from 4000 to 400 cm⁻¹ at a resolution of 4 cm⁻¹.

The morphology of the NanoSr loaded BGs as well as the development of hydroxyapatite layer on the surface of the NanoSr loaded BGs after soaking in SBF solution were assessed by means of scanning electron microscope (SEM) (SEM, TESCAN, Warrendale, PA). The dry powder of the sample were sputter-coated with gold before the examination.

2.3. Bioactivity evaluation test

To test the bioactivity of the samples, a simulated body fluid (SBF) including NaCl, KCl, MgCl₂·6H₂O and CaCl₂ was prepared. To this end, 50 mg of BGs and NanoSr loaded BGs were immersed in 40 ml of the SBF separately. Then, the solutions were incubated at 37ºC at a constant stirring of 100 rpm for 2 weeks. Lastly, the sample treated in SBF was washed with acetone to avoid more reaction. A scanning electron picture was applied to confirm the existence of the hydroxyapatite layer on the sample’ s surface.

3. Results and discussion

Different forms of bioactive glasses have been broadly tested so far owing to their outstanding potential in bone repair and regeneration [9]. Many recent reports have been on advance BGs with the better ability for induction osteogenesis and angiogenesis action in vitro and in vivo. Numerous methods have reported in this case and in most newly, some elements such as Sr is added to the basic building of glasses [21, 25]. Recent studies show a growing number of reports to use Sr in the production of permeable bone tissue engineering materials [26]. The XRD picks specified that the samples were in amorphous form after the remaining 2 weeks in the SBF solution.The data from the bioactivity examination in SBF showed the development of the hydroxyapatite layer on samples after 14 days. However, the hydroxyapatite formation was more for NanoSr loaded BGs. The gotten outcomes for some reports showed that the incorporation of Sr ion to the glass structure promotes its activity [10]. Bioactive glasses has shown the tendency to bond on bone tissue over the formation of a hydroxycarbonate apatite layer. This layer can stimulate osteogenesis action. Tarantino et al reported that Sr has a vital part in enhancing bone density and dropping fracture risk mammals [8]. In a work by Gentleman et al, Sr ions show ability to accelerate bone regeneration through increasing new bone deposition and decreasing bone resorption [10]. It has been suggested that ion-containing BGs can trigger angiogenesis-related genes through preparing a hypoxic condition and therefore are beneficial agents for bone tissue regeneration [14, 15].

Figure 1. XRD patterns of the: (a) BGs, (b) NanoSr loaded BGs

Figure 2. FTIR patterns of the: (a) BGs, (b) NanoSr loaded BGs

Figure 3. SEM of the BGs surface after immersion in SBF.