Smart Bone Graft Composite for Cancer Therapy Using Magnetic Hyperthermia.

Magnetic hyperthermia (MHT) is a therapy that uses the heat generated by a magnetic material for cancer treatment. Magnetite nanoparticles are the most used materials in MHT. However, magnetite has a high Curie temperature (Tc~580 °C), and its use may generate local superheating. To overcome this problem, strontium-doped lanthanum manganite could replace magnetite because it shows a Tc near the ideal range (42-45 °C). In this study, we developed a smart composite formed by an F18 bioactive glass matrix with different amounts of Lanthanum-Strontium Manganite (LSM) powder (5, 10, 20, and 30 wt.% LSM). The effect of LSM addition was analyzed in terms of sinterability, magnetic properties, heating ability under a magnetic field, and in vitro bioactivity. The saturation magnetization (Ms) and remanent magnetization (Mr) increased by the LSM content, the confinement of LSM particles within the bioactive glass matrix also caused an increase in Tc. Calorimetry evaluation revealed a temperature increase from 5 °C (composition LSM5) to 15 °C (LSM30). The specific absorption rates were also calculated. Bioactivity measurements demonstrated HCA formation on the surface of all the composites in up to 15 days. The best material reached 40 °C, demonstrating the proof of concept sought in this research. Therefore, these composites have great potential for bone cancer therapy and should be further explored.

Effect of bioactive Biosilicate® /F18 glass scaffolds on osteogenic differentiation of human adipose stem cells.

This study evaluated the gene expression profile of the human adipose-derived stem cells (hASCs) grown on the Biosilicate® /F18 glass (BioS-2P/F18) scaffolds. hASCs were cultured using the osteogenic medium (control), the scaffolds, and their ionic extract. We observed that ALP activity was higher in hASCs grown on the BioS-2P/F18 scaffolds than in hASCs cultured with the ionic extract or the osteogenic medium on day 14. Moreover, the dissolution product group and the control exhibited deposited calcium, which peaked on day 21. Gene expression profiles of cell cultured using the BioS-2P/F18 scaffolds and their extract were evaluated in vitro using the RT2 Profiler polymerase chain reaction (PCR) microarray on day 21. Mineralizing tissue-associated proteins, differentiation factors, and extracellular matrix enzyme expressions were measured using quantitative PCR. The gene expression of different proteins involved in osteoblast differentiation was significantly up-regulated in hASCs grown on the scaffolds, especially BMP1, BMP2, SPP1, BMPR1B, ITGA1, ITGA2, ITGB1, SMAD1, and SMAD2, showing that both the composition and topographic features of the biomaterial could stimulate osteogenesis. This study demonstrated that gene expression of hASCs grown on the scaffold surface showed significantly increased gene expression related to hASCs cultured with the ionic extract or the osteogenic medium, evidencing that the BioS-2P/F18 scaffolds have a substantial effect on cellular behavior of hASCs.

Putty-like bone fillers based on CaP ceramics or Biosilicate® combined with carboxymethylcellulose: Characterization, optimization, and evaluation.

Calcium phosphates and bioactive glass ceramics have been considered promising biomaterials for use in surgeries. However, their moldability should be further enhanced. We here thereby report the handling, physicochemical features, and morphological characteristics of formulations consisting of carboxymethylcellulose-glycerol and hydroxyapatite-tricalcium phosphate or Biosilicate® particles. We hypothesized that combining either material with carboxymethylcellulose-glycerol would improve handling properties, retaining their bioactivity. In addition to scanning electron microscopy, cohesion, mineralization, pH, and viscoelastic properties of the novel formulations, cell culture experiments were performed to evaluate the cytotoxicity and cell proliferation. Putty-like formulations were obtained with improved cohesion and moldability. Remarkably, mineralization in simulated body fluid of hydroxyapatite-tricalcium phosphate/carboxymethylcellulose-glycerol formulations was enhanced compared to pure hydroxyapatite-tricalcium phosphate. Cell experiments showed that all formulations were noncytotoxic and that HA-TCP60 and BGC50 extracts led to an increased cell proliferation. We conclude that combining carboxymethylcellulose-glycerol with either hydroxyapatite-tricalcium phosphate or Biosilicate® allows for the generation of moldable putties, improves handling properties, and retains the ceramic bioactivity.

Porous poly (D,L-lactide-co-glycolide) acid/biosilicate® composite scaffolds for bone tissue engineering.

This study evaluated the effects of the Biosilicate® and poly (D,L-lactic-co-glycolic) acid composites on bone repair in a tibial bone defect model in rats by means of using histological evaluation (histopathological and morphometric analysis) and gene expression analysis. Eighty male Wistar rats (12 weeks old, weighing ±300 g) were randomly divided into two groups: Biosilicate® group (BG) and Biosilicate® /PLGA group (BG/PLGA). Each group was euthanized at 3, 7, 14, and 21 days after surgery (n = 10 animals per time point). The main findings showed that the incorporation of PLGA into BG had a significant effect on the morphological structure of the material, accelerating mass loss, decreasing the pH and increasing the calcium release. Furthermore, histologic analysis revealed that the BG/PLGA showed increased material degradation, accompanied by higher bone formation compared to BG, after 21 days of implantation. In addition, qRT-PCR analysis showed that BG/PLGA induced an upregulation of the osteogenic genes related to BMP4, Runx2, ALP, and OC. These results show that the present BG/PLGA composite may be used as a bone graft for inducing bone repair. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 63-71, 2017.

Bioactive glass-based surfaces induce differential gene expression profiling of osteoblasts.

The ability of Biosilicate® with two crystalline phases (BioS-2P) to drive osteoblast differentiation encourages the investigation of the cellular mechanisms involved in this process. Then, the aim of our study was to analyze the large-scale gene expression of osteoblasts grown on BioS-2P compared with Bioglass® 45S5 (45S5). Osteoblasts differentiated from rat bone marrow mesenchymal stem cells were cultured under osteogenic conditions on BioS-2P, 45S5 and polystyrene (control). After 10 days, the expression of 23,794 genes was analyzed using mRNA Sequencing and the data were validated by real-time PCR. The BioS-2P exhibited 5 genes upregulated and 3 downregulated compared with 45S5. Compared with control, BioS-2P upregulated 15 and downregulated 11 genes, while 45S5 upregulated 25 and downregulated 21 genes. Eight genes were commonly upregulated and 4 downregulated by both bioactive glasses. In conclusion, our results demonstrated that bioactive glasses affect the gene expression profiling of osteoblasts. Most of the regulated genes by both BioS-2P and 45S5 are associated with the process of mineralization highlighting their osteostimulation property that is, at least in part, derived from the ability to modulate the intracellular machinery to promote osteoblast genotype expression. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 419-423, 2017.

Bone regeneration and gene expression in bone defects under healthy and osteoporotic bone conditions using two commercially available bone graft substitutes.

Biosilicate(®) and Bio-Oss(®) are two commercially available bone substitutes, however, little is known regarding their efficacy in osteoporotic conditions. The purpose of this study was to evaluate the osteogenic properties of both materials, at tissue and molecular level. Thirty-six Wistar rats were submitted to ovariectomy (OVX) for inducing osteoporotic conditions and sham surgery (SHAM) as a control. Bone defects were created in both femurs, which were filled with Biosilicate(®) or Bio-Oss(®), and empty defects were used as control. For the healthy condition both Biosilicate(®) and Bio-Oss(®) did not improve bone formation after 4 weeks. Histomorphometric evaluation of osteoporotic bone defects with bone substitutes showed more bone formation, significant for Bio-Oss(®). Molecular biological evaluation was performed by gene-expression analysis (Runx-2, ALP, OC, OPG, RANKL). The relative gene expression was increased with Biosilicate(®) for all genes in OVX rats and for Runx-2, ALP, OC and RANKL in SHAM rats. In contrast, with Bio-Oss(®), the relative gene expression of OVX rats was similar for all three groups. For SHAM rats it was increased for Runx-2, ALP, OC and RANKL. Since both materials improved bone regeneration in osteoporotic conditions, our results suggest that bone defects in osteoporotic conditions can be efficiently treated with these two bone substitutes.

Characterization and in vivo biological performance of biosilicate.

After an introduction showing the growing interest in glasses and glass-ceramics as biomaterials used for bone healing, we describe a new biomaterial named Biosilicate. Biosilicate is the designation of a group of fully crystallized glass-ceramics of the Na2O-CaO-SiO2-P2O5 system. Several in vitro tests have shown that Biosilicate is a very active biomaterial and that the HCA layer is formed in less than 24 hours of exposure to "simulated body fluid" (SBF) solution. Also, in vitro studies with osteoblastic cells have shown that Biosilicate disks supported significantly larger areas of calcified matrix compared to 45S5 Bioglass, indicating that this bioactive glass-ceramic may promote enhancement of in vitro bone-like tissue formation in osteogenic cell cultures. Finally, due to its special characteristics, Biosilicate has also been successfully tested in several in vivo studies. These studies revealed that the material is biocompatible, presents excellent bioactive properties, and is effective to stimulate the deposition of newly formed bone in animal models. All these data highlight the huge potential of Biosilicate to be used in bone regeneration applications.

Bioactivity and cytotoxicity of glass and glass-ceramics based on the 3CaO·P2O5--SiO2--MgO system.

The mechanical strength of bioactive glasses can be improved by controlled crystallization, turning its use as bulk bone implants viable. However, crystallization may affect the bioactivity of the material. The aim of this study was to develop glass-ceramics of the nominal composition (wt%) 52.75(3CaO·P2O5)-30SiO2-17.25MgO, with different crystallized fractions and to evaluate their in vitro cytotoxicity and bioactivity. Specimens were heat-treated at 700, 775 and 975 °C, for 4 h. The major crystalline phase identified was whitlockite, an Mg-substituted tricalcium phosphate. The evaluation of the cytotoxicity was carried out by the neutral red uptake methodology. Ionic exchanges with the simulated body fluid SBF-K9 acellular solution during the in vitro bioactivity tests highlight the differences in terms of chemical reactivity between the glass and the glass-ceramics. The effect of crystallinity on the rates of hydroxycarbonate apatite (HCA) formation was followed by Fourier transformed infrared spectroscopy. Although all glass-ceramics can be considered bioactive, the glass-ceramic heat-treated at 775 °C (V775-4) presented the most interesting result, because the onset for HCA formation is at about 24 h and after 7 days the HCA layer dominates completely the spectrum. This occurs probably due to the presence of the whitlockite phase (3(Ca,Mg)O·P2O5). All samples were considered not cytotoxic.

Histopathological, cytotoxicity and genotoxicity evaluation of Biosilicate® glass-ceramic scaffolds.

This study evaluated the biocompatibility of Biosilicate® scaffolds by means of histopathological, cytotoxicity, and genotoxicity analysis. The histopathologic analysis of the biomaterial was performed using 65 male rats, distributed into the groups: control and Biosilicate®, evaluated at 7, 15, 30, 45, and 60 days after implantation. The cytotoxicity analysis was performed by the methyl thiazolyl tetrazolium (MTT) assay, with various concentrations of extracts from the biomaterial in culture of osteoblasts and fibroblasts after 24, 72, and 120 h. The genotoxicity analysis (comet assay) was performed in osteoblasts and fibroblasts after contact with the biomaterial during 24, 72, and 96 h. In the histopathology analysis, we observed a foreign body reaction, characterized by the presence of granulation tissue after 7 days of implantation of the biomaterial, and fibrosis connective tissue and multinucleated giant cells for longer periods. In the cytotoxicity analysis, extracts from the biomaterial did not inhibit the proliferation of osteoblasts and fibroblasts, and relatively low concentrations (12.5% and 25%) stimulated the proliferation of both cell types after 72 and 120 h. The analysis of genotoxicity showed that Biosilicate® did not induce DNA damage in both lineages tested in all periods. The results showed that the Biosilicate® scaffolds present in vivo and in vitro biocompatibility.

Biosilicate®-gelatine bone scaffolds by the foam replica technique: development and characterization.

The development of bioactive glass-ceramic materials has been a topic of great interest aiming at enhancing the mechanical strength of traditional bioactive scaffolds. In the present study, we test and demonstrate the use of Biosilicate® glass-ceramic powder to fabricate bone scaffolds by the foam replica method. Scaffolds possessing the main requirements for use in bone tissue engineering (95% porosity, 200-500 µm pore size) were successfully produced. Gelatine coating was investigated as a simple approach to increase the mechanical competence of the scaffolds. The gelatine coating did not affect the interconnectivity of the pores and did not significantly affect the bioactivity of the Biosilicate® scaffold. The gelatine coating significantly improved the compressive strength (i.e. 0.80 ± 0.05 MPa of coated versus 0.06 ± 0.01 MPa of uncoated scaffolds) of the Biosilicate® scaffold. The combination of Biosilicate® glass-ceramic and gelatine is attractive for producing novel scaffolds for bone tissue engineering.