Gene activation by bioactive glasses

Bioactive glasses have been shown to regulate gene expression in both hard and soft tissue repair. New resorbable bioactive glass constructs are now being developed that can influence gene expression in the local environment by manipulating material properties such as the surface chemistry, topography and the release of dissolution ions. The success of these scaffolds, however, may depend upon a greater understanding of the bioactive glass stimulated gene expression pathways. This will allow the construction of tissue specific scaffolds with tailored surface chemistry, topography and ion release rates. This paper summarises the advances made in understanding gene expression in response to bioactive glasses and discusses the future steps required for further insights into these molecular mechanisms.     

Interaction between bioactive glasses and human dentin

This study explores the interaction between bioactive glasses and dentin from extracted human teeth in simulated oral conditions. Bioactive glasses in the Na₂O–CaO–P₂O₅–SiO₂ and MgO–CaO–P₂O₅–SiO₂ systems were prepared as polished disks. Teeth were prepared by grinding to expose dentin and etching with phosphoric acid. A layer of saliva was placed between the two, and the pair was secured with an elastic band and immersed in saliva at 37 °C for 5, 21 or 42 days. The bioactive glasses adhered to dentin, while controls showed no such interaction. A continuous interface between the bioactive glass and dentin was imaged using cryogenic-scanning electron microscopy (SEM). However, after alcohol dehydration and critical point drying, fracture occurred due to stresses from dentin shrinkage. SEM investigations showed a microstructurally different material at the fractured interface. Chemical analyses revealed that ions from the glass penetrated into the dentin and that the surface of the glass in contact with the dentin was modified. Microdiffractometry showed the presence of apatite at the interface. Bonding appears to be due to an affinity of collagen for the glass surface and chemical interaction between the dentin and glass, leading to apatite formation at the interface.     

Angiogenic potential of boron-containing bioactive glasses: in vitro study

Boron-containing bioactive glasses (BGs) are being extensively researched for the treatment and regeneration of bone defects because of their osteostimulatory and neovascularization potential. In this study, we report the effects of the ionic dissolution products (IDPs) of different boron-doped, borosilicate, and borate BG scaffolds on mouse bone marrow stromal cells in vitro, using an angiogenesis assay. Five different BG scaffolds of the system SiO₂–Na₂O–K₂O–MgO–CaO–P₂O₅–B₂O₃ (with varying amounts of SiO₂ and B₂O₃) were fabricated by the foam replication technique. Bone marrow stromal cells were cultivated in contact with the IDPs of the boron-containing BG scaffolds at different concentrations for 48 h. The expression and secretion of vascular endothelial growth factor (VEGF) from the cultured cells was measured quantitatively using the VEGF ELISA Kit. Cell viability and cell morphology were determined using WST-8 assay and H&E staining, respectively. The cellular response was found to be dependent on boron content and the B release profile from the glasses corresponded to the positive or negative biological activity of the BGs.     

Early stage reactivity and in vitro behavior of silica-based bioactive glasses and glass-ceramics

The surface reactivity of different sets of glasses and glass-ceramics belonging to the SiO₂–P₂O₅–CaO–MgO–K₂O–Na₂O system have been investigated. The attention was focused on the role of their composition on the bioactivity kinetics, in terms of pH modifications, silica-gel formation and its evolution toward hydroxycarbonatoapatite, after different times of soaking in simulated body fluid. Glasses and glass ceramics have been characterized by thermal analysis, SEM-EDS observations and phase analysis (XRD). XPS measurements have been carried out on the most representative set of sample in order to evaluate the evolution of the surface species during the growth of silica-gel and hydroxycarbonatoapatite. The response of murine fibroblast 3T3 to the material before and after a conditioning pre-treatment (immersion in SBF) has been investigated on the same set of samples in order to point out the role of the bioactivity mechanism on cell viability. The main differences among the various glasses have been related to the modifier oxides ratio and to the MgO content, which seems to have an influence on the glass stability, both in terms of thermal properties and surface reactivity. The surface characterization and in vitro tests revealed few variations in the reactivity of the different glasses and glass-ceramics in their pristine form. On the contrary, the different surface properties before and after the pre-treatment in SBF seem to play a role on the biocompatibility of both glass and glass-ceramics, due to the different ion release and hydrophilicity of the surfaces, affecting both cell viability and protein adsorption.     

Cytotoxicity assessment of modified bioactive glasses with MLO-A5 osteogenic cells in vitro

The primary objective of this study was to evaluate in vitro responses of MLO-A5 osteogenic cells to two modifications of the bioactive glass 13-93. The modified glasses, which were designed for use as cell support scaffolds and contained added boron to form the glasses 13-93 B1 and 13-93 B3, were made to accelerate formation of a bioactive hydroxyapatite surface layer and possibly enhance tissue growth. Quantitative MTT cytotoxicity tests revealed no inhibition of growth of MLO-A5 cells incubated with 13-93 glass extracts up to 10 mg/ml, moderate inhibition of growth with 13-93 B1 glass extracts, and noticeable inhibition of growth with 13-93 B3 glass extracts. A morphology-based biocompatibility test was also performed and yielded qualitative assessments of the relative biocompatibilities of glass extracts that agree with those obtained by the quantitative MTT test. However, as a proof of concept experiment, when MLO-A5 cells were seeded onto 13-93 B3 scaffolds in a dynamic in vitro environment, cell proliferation occurred as evidenced by qualitative and quantitative MTT labeling of scaffolds. Together these results demonstrate the in vitro toxicity of released borate ion in static experiments; however borate ion release can be mitigated in a dynamic environment similar to the human body where microvasculature is present. Here we argue that despite toxicity in static environments, boron-containing 13-93 compositions may warrant further study for use in tissue engineering applications.     

In vitro behaviour of three biocompatible glasses in composite implants

Poly(l,dl-lactide) composites containing filler particles of bioactive glasses 45S5 and S53P4 were compared with a composite containing a slowly dissolving glass S68. The in vitro reactivity of the composites was studied in simulated body fluid, Tris-buffered solution, and phosphate buffered saline. The high processing temperature induced thermal degradation giving cavities in the composites containing 45S5 and S53P4, while good adhesion of S68 to the polymer was observed. The cavities partly affected the in vitro reactivity of the composites. The degradation of the composites containing the bioactive glasses was faster in phosphate buffered saline than in the two other solutions. Hydroxyapatite precipitation suggesting bone tissue bonding capability was observed on these two composites in all three solutions. The slower dissolution of S68 glass particles and the limited hydroxyapatite precipitation suggested that this glass has potential as a reinforcing composition with the capability to guide bone tissue growth in biodegradable polymer composites.     

Alkali oxide containing mesoporous bioactive glasses: Synthesis,characterization and in vitro bioactivity

We report, for the first time, the synthesis of sodium oxide containing mesoporous bioactive quaternary glasses and compared with two different mesoporous ternary silicate systems by modified sol–gel process. With the aid of three different glass systems, a systematic analysis has been made on phosphorous-bearing (P-bearing) and phosphorous-free (P-free) mesoporous bioactive glasses to investigate the role of phosphorus on in vitro bioactivity of various silicate glasses with constant alkali oxide content. The combined use of multiple analytical techniques XRD, FTIR, SEM, nitrogen adsorption/desorption analysis before and after soaking in the SBF solution allowed us to establish strong correlation between composition, pore structure and bioactivity. We find that the P-bearing mesoporous glasses show the rapid hydroxycarbonate apatite (HCA) crystallization than P-free mesoporous glasses independent of calcium content. The present study reveals that the presence of phosphorous jointly with calcium in the bioactive glass system significantly enhances the rate of apatite formation as well as crystallization of apatite phase. Additionally, we find that a glass with sodium orthophosphate rich phase enhances the solubility when immersed in SBF and further accelerate the kinetics of apatite formation. The influences of the chemical composition and their superior textural properties on bioactivity are explained in terms of the unique structure of mesoporous bioactive glasses.     

In vitro fluoride uptake by bovine enamel in using cyanoacrylate adhesives containing fluoride compounds

To study thein vitro uptake of fluoride from 3-year-old bovine enamel, enamel biopsies for fluoride analysis were performed using five successive etchings. At 10 µm etch depth the uptake of fluoride was maximum for all methyl--cyanoacrylates containing six kinds of fluoride compounds. The fluoride concentration was pronounced by 1 month of immersion, rather than by 1 week of immersion in distilled water. In particular, fluoride compounds such as BiF₃, NaF, SnF₂ and ZnF₂ had a significant difference (p<0.01) from KF and Na₂FPO₄ at 1 month of immersion.     

生物玻璃的原位复合及其生物活性

以正硅酸乙酯(TEOS)、磷酸三乙酯(TEP)、四水硝酸钙为无机前驱体,采用溶胶凝胶法原位复合聚乙烯醇(PVA)制备钙磷硅生物玻璃,研究了工艺流程、无机相前驱体的水解时间及模板的使用等对生物玻璃性能的影响.结果表明,在生物玻璃的原位复合中PVA模板与钙磷硅前驱体混合的工艺顺序不同,则样品的韧性不同.600℃热处理去除有机模板得到的生物的玻璃粉体表现出较高的比表面积和较大的中孔体积,在体外生理模拟液中浸泡7 d后矿化形成的活性碳酸钙与羟基磷灰石(HAP)微晶具有较高的生物活性.     

Thermal behaviour of bioactive alkaline-earth silicophosphate glasses

The thermal behaviour (sinterability and first crystallization) of a series of alkaline-earth silicophosphate glasses has been studied by differential thermal analysis (DTA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). The samples were prepared from a base bioactive glass of the system CaO–P₂O₅–SiO₂–CaF₂, by (a) slightly changing the F/O ratio; (b) replacing part of the CaO by SrO or MgO; and (c) increasing the total alkaline earth concentration by MgO additions. The results show that the addition of MgO is the most effective way of improving sinterability. In these samples, a decrease of the glass transition temperature, together with an increase in the temperature of the first crystallization, is observed. The difference between both temperatures is proposed to be an adequate indicator of the sinterability. The initial stages of the first crystallization (which produces an oxo-fluorapatite), and its composition dependence, are discussed in terms of the results of sinterability, and the classical theory of nucleation.     

In vitro evaluation of borate-based bioactive glass scaffolds prepared by a polymer foam replication method

Borate-based bioactive glass scaffolds with a microstructure similar to that of human trabecular bone were prepared using a polymer foam replication method, and evaluated in vitro for potential bone repair applications. The scaffolds (porosity = 72 ± 3%; pore size = 250–500 μm) had a compressive strength of 6.4 ± 1.0 MPa. The bioactivity of the scaffolds was confirmed by the formation of a hydroxyapatite (HA) layer on the surface of the glass within 7 days in 0.02 M K₂HPO₄ solution at 37 °C. The biocompatibility of the scaffolds was assessed from the response of cells to extracts of the dissolution products of the scaffolds, using assays of MTT hydrolysis, cell viability, and alkaline phosphatase activity. For boron concentrations below a threshold value (0.65 mM), extracts of the glass dissolution products supported the proliferation of bone marrow stromal cells, as well as the proliferation and function of murine MLO-A5 cells, an osteogenic cell line. Scanning electron microscopy showed attachment and continuous increase in the density of MLO-A5 cells cultured on the surface of the glass scaffolds. The results indicate that borate-based bioactive glass could be a potential scaffold material for bone tissue engineering provided that the boron released from the glass could be controlled below a threshold value.     

Influence of calcium and phosphorus release from bioactive glasses on viability and differentiation of dental pulp stem cells

The release of ions that can significantly contribute toward cellular response is an important characteristic of bioactive glasses (BG). Here, ionic extracts of three different compositions of BG powders in 60 mol% SiO₂, x mol% CaO (x = 28, 32 and 36), x mol% P₂O₅ (x = 12, 8 and 4) compositional system were utilized to study their effect on the viability, differentiation and mineralization of dental pulp stem cells (DPSCs) in vitro. ICP was applied to detect the exact ionic concentrations released from different composition of BG. DPSCs treated with conditioned media from the glass with 4 mol% P₂O₅ (BGCM1, media containing 44.01 ± 0.6 mg/L Si, 61.72 ± 0.1 mg/L Ca and 7.57 ± 0.01 mg/L P) were more metabolically active compared to conditioned media from the glass with 8 mol% P₂O₅ (BGCM2, media with 47.36 ± 0.7 mg/L Si, 57.4 ± 0.1 mg/L Ca and 14.54 ± 0.2 mg/L P), at all times tested, but in all cases the process was slower than the control. Cells exposed to media conditioned by the glass with 12 mol% P₂O₅ (BGCM3, 40.46 ± 0.5 mg/L Si, 61. 85 ± 0.3 mg/L Ca and 28.43 ± 0.3 mg/L P) responded differently, such that cells showed to be more metabolically active than control at day 3, but then similar to or lower than control at higher time points. Differentiation of DPSCs toward osteogenic lineage in the presence of BGCM was assessed by Alizarin red staining. Cells treated with high phosphate BGCM3 displayed a higher density of red mineralized nodules than cells treated with BGCM1 and BGCM2 after 21 days of culture in non-osteogenic medium. BGCM3 was therefore chosen for gene expression studies. Osteogenic differentiation of DPSCs in the presence and/or absence of BGCM3 or osteogenic supplements were studied by RT-PCR. Overall, the results demonstrated that, in the absence of osteogenic supplements, BGCM3 group showed a significantly higher mRNA expression levels for alkaline phosphatase at day 7, osteopontin and osteonectin at days 7 and 14, and a high level of collagen I at day 14, compared to negative control group (BM?). Overall, the results obtained from BGCM3 group are beneficial for the design and manufacture of scaffolds or particulates with tailored ion release for a range of bone repair applications.     

In vitro evaluation of gentamicin release from a bioactive tricalcium silicate bone cement

Tricalcium silicate (Ca₃SiO₅) cement, a novel self-setting biomaterial, has been shown to exhibit good hydraulic properties and excellent bioactivity. In this study, gentamicin sulfate (GS) was integrated into cement pastes and in vitro release of GS from the Ca₃SiO₅ cement was performed in deionized water, phosphate buffer saline (PBS) and HCl solutions with different pH at 37 °C, respectively. The results showed that the initial fast release of GS was restricted to a low level and prolonged release of drugs was achieved in water and PBS. The prolonged GS release is attributed to the interaction of GS with the calcium silicate hydrate network and the formation of unique nano-to-micro porous structure after hydration. Furthermore, GS release from milled powders of the hydrated cement suggested that the constrained GS could be released at low pH environment or during the degradation of the cement. When the samples were soaked in PBS, a nano-structured apatite layer was formed on the surface of the cement, which resulted in a relatively lower GS release rate as compared to that in water. The results suggest that Ca₃SiO₅ cement might be used as bioactive bone implant materials with drug loading and prolonged release properties.     

Bactericidal effects of bioactive glasses on clinically important aerobic bacteria

Bioactive glasses (BAGs) have been studied for decades for clinical use, and they have found many dental and orthopedic applications. BAGs have also been shown to have an antibacterial effect e.g., on some oral microorganisms. In this extensive work we show that six powdered BAGs and two sol-gel derived materials have a clear antibacterial effect on 29 clinically important bacterial species. We also incorporated a rapid and accurate flow cytometric (FCM) method to calculate and standardize the numbers of viable bacteria inoculated in the suspensions used in the tests for antibacterial activity. In all materials tested growth inhibition could be demonstrated, although the concentration and time needed for the effect varied depending on the BAG. The most effective glass was S53P4, which had a clear growth-inhibitory effect on all pathogens tested. The sol-gel derived materials CaPSiO and CaPSiO II also showed a strong antibacterial effect. In summary, BAGs were found to clearly inhibit the growth of a wide selection of bacterial species causing e.g., infections on the surfaces of prostheses in the body after implantation.     

Effects of hydrolysis on dodecyl alcohol-modified bioactive glasses and PDLLA/modified bioactive glass composite films

In this article, mesoporous 58S and 58S bioactive glasses (BGs) were surface modified by dodecyl alcohol through esterification reaction and PDLLA/modified BGs composite films were prepared. The purpose of this study was to investigate the properties of the modified BGs particles and the PDLLA/modified BGs composite films before and after hydrolytic treatment. The modified BGs powders and composite films were treated in boiling water for 20 min to remove the dodecyl chains. After hydrolytic treatment, the modified BGs powders showed increased hydrophilicity and the FTIR analysis revealed that most dodecyl chains were removed. Furthermore, the hydrophilicity of the PDLLA/modified BGs composite films was also greatly improved. The tensile strength of the composite films after hydrolysis decreased slightly, but was still much higher than that of pure PDLLA film. In addition, bone marrow mesenchymal stem cells from dogs on the composite films after hydrolytic treatment showed the highest proliferation rate. The results suggest that hydrolytic treatment is an effective and practicable method to remove alcohol chains from surface-modified BGs and polymers/modified BG composites, which may be used for preparation of bioactive scaffolds for tissue engineering applications.     

Local structures of mesoporous bioactive glasses and their surface alterations in vitro: inferences from solid-state nuclear magnetic resonance

We review the benefits of using (29)Si and (1)H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy for probing the local structures of both bulk and surface portions of mesoporous bioactive glasses (MBGs) of the CaO-SiO(2)-(P(2)O(5)) system. These mesoporous materials exhibit an ordered pore arrangement, and are promising candidates for improved bone and tooth implants. We discuss experimental MAS NMR results from three MBGs displaying different Ca, Si and P contents: the (29)Si NMR spectra were recorded either directly by employing radio-frequency pulses to (29)Si, or by magnetization transfers from neighbouring protons using cross polarization, thereby providing quantitative information about the silicate speciation present in the pore wall and at the MBG surface, respectively. The surface modifications were monitored for the three MBGs during their immersion in a simulated body fluid (SBF) for intervals between 30 min and one week. The results were formulated as a reaction sequence describing the interconversions between the distinct silicate species. We generally observed a depletion of Ca(2+) ions at the MBG surface, and a minor condensation of the silicate-surface network over one week of SBF soaking.     

Development of soluble glasses for biomedical use Part I: In vitro solubility measurement

In this study soluble glasses have been developed for biomedical applications containing P₂O₅ as a network former and CaO and Na₂O as modifiers. This study shows that as expected, the glasses have an inverse exponential relationship between solubility and CaO content. Furthermore, there is evidence for compositional related non-linearity in the dissolution of the glasses with time. This is thought to be due to either layer formation on the glass surface hindering ion diffusion, ion exchange process or change of ionic strength of the solution. Bioactivity of these glasses is indicated by the formation of a brushite precipitate, a precursor to apatite formation. Further evidence for bioactivity is also presented in the second part of this paper.     

Calcium and potassium addition to facilitate the sintering of bioactive glasses

Nowadays bioactive glasses are diffused in medical practice due to their excellent bioactivity. However high temperature treatments, which are commonly required in several processing routes, may induce the glass to crystallize into a glass-ceramic, with possible negative effects on its bioactivity. In this work a new bioactive glass composition, inspired by the widely used Bioglass® 45 S5, was formulated by increasing the calcium content and substituting the sodium oxide with potassium oxide. The novel glass can be treated at a relatively low temperature (800 °C) and it is characterized by a reduced tendency to crystallize with excellent effects in terms of bioactivity, according to in vitro tests. Therefore, the new composition opens intriguing scenarios whenever a thermal treatment is required to apply or to sinter the glass, such as in the production of scaffolds or the deposition of coatings.