Interaction of monodispersed strontium containing bioactive glass nanoparticles with macrophages.

The cellular response of murine primary macrophages to monodisperse strontium containing bioactive glass nanoparticles (SrBGNPs), with diameters of 90 ± 10 nm and a composition (mol%) of 88.8 SiO2-1.8CaO-9.4SrO (9.4% Sr-BGNPs) was investigated for the first time. Macrophage response is critical as applications of bioactive nanoparticles will involve the nanoparticles circulating in the blood stream and macrophages will be the first cells to encounter the particles, as part of inflammatory response mechanisms. Macrophage viability and total DNA measurements were not decreased by particle concentrations of up to 250 µg/mL. The Sr-BGNPs were actively internalised by the macrophages via formation of endosome/lysosome-like vesicles bordered by a membrane inside the cells. The Sr-BGNPs degraded inside the cells, with the Ca and Sr maintained inside the silica network. When RAW264.7 cells were incubated with Sr-BGNPs, the cells were polarised towards the pro-regenerative M2 population rather than the pro-inflammatory M1 population. Sr-BGNPs are potential biocompatible vehicles for therapeutic cation delivery for applications in bone regeneration.

Scaffold channel size influences stem cell differentiation pathway in 3-D printed silica hybrid scaffolds for cartilage regeneration.

We report that 3-D printed scaffold channel size can direct bone marrow derived stem cell differentiation. Treatment of articular cartilage trauma injuries, such as microfracture surgery, have limited success because durability is limited as fibrocartilage forms. A scaffold-assisted approach, combining microfracture with biomaterials has potential if the scaffold can promote articular cartilage production and share load with cartilage. Here, we investigated human bone marrow derived stromal cell (hBMSC) differentiation in vitro in 3-D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) hybrid scaffolds with specific channel sizes. Channel widths of ∼230 µm (210 ± 22 µm mean strut size, 42.4 ± 3.9% porosity) provoked hBMSC differentiation down a chondrogenic path, with collagen Type II matrix prevalent, indicative of hyaline cartilage. When pores were larger (∼500 µm, 229 ± 29 µm mean strut size, 63.8 ± 1.6% porosity) collagen Type I was dominant, indicating fibrocartilage. There was less matrix and voids in smaller channels (∼100 µm, 218 ± 28 µm mean strut size, 31.2 ± 2.9% porosity). Our findings suggest that a 200-250 µm pore channel width, in combination with the surface chemistry and stiffness of the scaffold, is optimal for cell-cell interactions to promote chondrogenic differentiation and enable the chondrocytes to maintain their phenotype.

Electrospinning 3D bioactive glasses for wound healing.

An electrospinning technique was used to produce three-dimensional (3D) bioactive glass fibrous scaffolds, in the SiO2-CaO sol-gel system, for wound healing applications. Previously, it was thought that 3D cotton wool-like structures could only be produced from sol-gel when the sol contained calcium nitrate, implying that the Ca2+ and its electronic charge had a significant effect on the structure produced. Here, fibres with a 3D appearance were also electrospun from compositions containing only silica. A polymer binding agent was added to inorganic sol-gel solutions, enabling electrospinning prior to bioactive glass network formation and the polymer was removed by calcination. While the addition of Ca2+ contributes to the 3D morphology, here we show that other factors, such as relative humidity, play an important role in producing the 3D cotton-wool-like macrostructure of the fibres. A human dermal fibroblast cell line (CD-18CO) was exposed to dissolution products of the samples. Cell proliferation and metabolic activity tests were carried out and a VEGF ELISA showed a significant increase in VEGF production in cells exposed to the bioactive glass samples compared to control in DMEM. A novel SiO2-CaO nanofibrous scaffold was created that showed tailorable physical and dissolution properties, the control and composition of these release products are important for directing desirable wound healing interactions.

Osteogenic potential of sol-gel bioactive glasses containing manganese.

Bioactive glasses (BGs) are widely used for bone regeneration, and allow the incorporation of different ions with therapeutic properties into the glass network. Amongst the different ions with therapeutic benefits, manganese (Mn) has been shown to influence bone metabolism and activate human osteoblasts integrins, improving cell adhesion, proliferation and spreading. Mn has also been incorporated into bioceramics as a therapeutic ion for improved osteogenesis. Here, up to 4.4 mol% MnO was substituted for CaO in the 58S composition (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) and its effects on the glass properties and capability to influence the osteogenic differentiation were evaluated. Mn-containing BGs with amorphous structure, high specific surface area and nanoporosity were obtained. The presence of Mn2+ species was confirmed by X-ray photoelectron spectroscopy (XPS). Mn-containing BGs presented no cytotoxic effect on human mesenchymal stem cells (hMSCs) and enabled sustained ion release in culture medium. hMSCs osteogenic differentiation stimulation and influence on the mineralisation process was also confirmed through the alkaline phosphatase (ALP) activity, and expression of osteogenic differentiation markers, such as collagen type I, osteopontin and osteocalcin, which presented higher expression in the presence of Mn-containing samples compared to control. Results show that the release of manganese ions from bioactive glass provoked human mesenchymal stem cell (hMSC) differentiation down a bone pathway, whereas hMSCs exposed to the Mn-free glass did not differentiate. Mn incorporation offers great promise for obtaining glasses with superior properties for bone tissue regeneration.

Effects of manganese incorporation on the morphology, structure and cytotoxicity of spherical bioactive glass nanoparticles.

Bioactive glass nanoparticles (BGNPs) are of great interest in tissue engineering as they possess high dissolution rate and capability of being internalized by cells, releasing their dissolution products with therapeutic benefits intracellularly. A modified Stöber process can be applied to obtain different BGNPs compositions containing therapeutic ions while maintaining controllable particle morphology, monodispersity and reduce agglomeration. Here, BGNPs containing Mn, an ion that has been shown to influence the osteoblast proliferation and bone mineralization, were evaluated. Particles with up to 142.3 ±â€¯10.8 nm and spherical morphology were obtained after MnO incorporation in the SiO2 - CaO system. X-ray photoelectron spectroscopy (XPS) indicated the presence of Mn2+ species and also a reduction in the number of bridging oxygen bonds due to the Ca and Mn. The Ca and Mn network modifier role on the silica network was also confirmed by magic-angle spinning 29Si solid-state nuclear magnetic resonance (MAS NMR). MTT evaluation showed no reduction in the mitochondrial metabolic activity of human mesenchymal stem cells exposed to the glass ionic products. Thus, evaluation showed that Mn could be incorporated into BGNPs by the modified Stöber method while maintaining their spherical morphology and features as a promising strategy for tissue regeneration.

Human mesenchymal stem cells differentiate into an osteogenic lineage in presence of strontium containing bioactive glass nanoparticles.

While bioactive glass and ions released during its dissolution are known to stimulate osteoblast cells, the effect bioactive glass has on human stem cells is not clear. Here, we show that spherical monodispersed strontium containing bioactive nanoparticles (Sr-BGNPs) of composition 90.6 mol% SiO2, 5.0 mol% CaO, 4.4% mol% SrO (4.4%Sr-BGNPs) and 88.8 mol% SiO2, 1.8 mol% CaO, and 9.4 mol% SrO (9.4%Sr-BGNPs) stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without osteogenic supplements. The particles were synthesised using a modified StÓ§ber process and had diameters of 90 ±â€¯10 nm. Previous work on similar particles that did not contain Sr (80 mol% SiO2, 20 mol% CaO) showed stem cells did not differentiate when exposed to the particles. Here, both compositions of the Sr-BGNPs (up to concentration of 250 µg/mL) stimulated the early-, mid-, and late-stage markers of osteogenic differentiation and accelerated mineralisation in the absence of osteogenic supplements. Sr ions play a key role in osteogenic stem cell differentiation. Sr-BGNP dissolution products did not adversely affect hMSC viability and no significant differences in viability were measured between each particle composition. Confocal and transmission electron microscopy (TEM) demonstrated that monodispersed Sr-BGNPs were internalised and localised within vesicles in the cytoplasm of hMSCs. Degradation of particles inside the cells was observed, whilst maintaining effective cations (Ca and Sr) in their silica network after 24 h in culture. The uptake of Sr-BGNPs by hMSCs was reduced by inhibitors of specific routes of endocytosis, indicating that the Sr-BGNPs uptake by hMSCs was probably via mixed endocytosis mechanisms. Sr-BGNPs have potential as injectable therapeutic devices for bone regeneration or treatment of conditions such as osteoporosis, because of their ability deliver a sustained release of osteogenic inorganic cations, e.g. calcium (Ca) or and strontium (Sr), through particle degradation locally to cells. STATEMENT OF SIGNIFICANCE: Here, we show that 90 nm spherical strontium containing bioactive nanoparticles of stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without the use of osteogenic supplements. While bioactive glass and its dissolution products are known to promote excellent bone regeneration in vivo and to stimulate osteoblast cells to produce bone matrix in vitro, their effect on human stem cells is not clear. Previously our nanoparticles that contained only SiO2 and CaO did not provoke human bone marrow or adipose derived stem cell differentiation.

Silk fibroin-bioactive glass based advanced biomaterials: towards patient-specific bone grafts.

A major challenge in bone tissue engineering is to develop patient-specific, defect-site specific grafts capable of triggering specific cell signaling pathways. We could programmably fabricate the 3D printed bone constructs via direct ink writing of silk-gelatin-bioactive glass (SF-G-BG) hybrids using two different compositions of melt-derived bioactive glasses (with and without strontium) and compared against commercial 45S5 Bioglass®. Physico-chemical characterization revealed that released ions from bioactive glasses inhibited the conformational change of Bombyx mori silk fibroin protein (from random coil to ß-sheet conformation), affecting printability of the SF-G-BG ink. In-depth molecular investigations showed that strontium containing SF-G-BG constructs demonstrated superior osteogenic differentiation of mesenchymal stem cells (TVA-BMSCs) over 21 days towards osteoblastic (marked by upregulated expression of runt related transcription factor, alkaline phosphatase, osteopontin, osteonectin, integrin bone sialoprotein, osteocalcin) and osteocytic (marked by podoplanin, dentin matrix acidic phosphoprotein, sclerostin) phenotype compared to other BG compositions and silk-gelatin alone. Moreover, ionic release from bioactive glasses in the silk-gelatin ink triggered the activation of signaling pathways (BMP-2, BMP-4 and IHH), which are critical in regulating bone formation in vivo. Overall, the presence of strontium containing bioactive glass in silk-gelatin matrices provided appropriate cues in regulating the development of custom-made 3D in vitro human bone constructs.

Toward smart implant synthesis: bonding bioceramics of different resorbability to match bone growth rates.

Craniofacial reconstructive surgery requires a bioactive bone implant capable to provide a gradual resorbability and to adjust to the kinetics of new bone formation during healing. Biomaterials made of calcium phosphate or bioactive glasses are currently available, mainly as bone defect fillers, but it is still required a versatile processing technique to fabricate composition-gradient bioceramics for application as controlled resorption implants. Here it is reported the application of rapid prototyping based on laser cladding to produce three-dimensional bioceramic implants comprising of a calcium phosphate inner core, with moderate in vitro degradation at physiological pH, surrounded by a bioactive glass outer layer of higher degradability. Each component of the implant is validated in terms of chemical and physical properties, and absence of toxicity. Pre-osteoblastic cell adhesion and proliferation assays reveal the adherence and growth of new bone cells on the material. This technique affords implants with gradual-resorbability for restoration of low-load-bearing bone.

Theranostic mesoporous silica nanoparticles biodegrade after pro-survival drug delivery and ultrasound/magnetic resonance imaging of stem cells.

Increasing cell survival in stem cell therapy is an important challenge for the field of regenerative medicine. Here, we report theranostic mesoporous silica nanoparticles that can increase cell survival through both diagnostic and therapeutic approaches. First, the nanoparticle offers ultrasound and MRI signal to guide implantation into the peri-infarct zone and away from the most necrotic tissue. Second, the nanoparticle serves as a slow release reservoir of insulin-like growth factor (IGF)-a protein shown to increase cell survival. Mesenchymal stem cells labeled with these nanoparticles had detection limits near 9000 cells with no cytotoxicity at the 250 µg/mL concentration required for labeling. We also studied the degradation of the nanoparticles and showed that they clear from cells in approximately 3 weeks. The presence of IGF increased cell survival up to 40% (p<0.05) versus unlabeled cells under in vitro serum-free culture conditions.

Hypoxia inducible factor-stabilizing bioactive glasses for directing mesenchymal stem cell behavior.

Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, proliferation, and recruitment to sites of injury. Materials capable of affecting the MSC oxygen-sensing pathway, independently of the environmental oxygen pressure, are therefore of immense interest to the tissue engineering (TE) and regenerative medicine community. In this study, we describe the evaluation of the effect of hypoxia inducible factor (HIF)-stabilizing bioactive glasses (BGs) on human MSCs. The dissolution products from these hypoxia-mimicking BGs stabilized HIF-1α in a concentration-dependent manner, altered cell proliferation and metabolism, and upregulated a number of genes involved in the hypoxic response (HIF1A, HIF2A, and VHL), MSC survival (SAG and BCL2), extracellular matrix remodeling (MMP1), and angiogenesis (VEGF and PDGF). These HIF-stabilizing materials can therefore be used to improve MSC survival and enhance regeneration in a number of TE strategies.

Monodispersed bioactive glass submicron particles and their effect on bone marrow and adipose tissue-derived stem cells.

Spherical monodispersed bioactive particles are potential candidates for nanocomposite synthesis or as injectable particles that could be internalized by cells for the local sustained delivery of inorganic therapeutic ions (e.g., calcium or strontium). Particles are also likely to be released from porous bioactive glass and sol-gel hybrid scaffolds as they degrade; thus, it is vital to investigate their interaction with cells. Spherical monodispersed bioactive glass particles (mono-SMBG), with diameters of 215 ± 20 nm are synthesized using a modified Stöber process. Confocal and transmission electron microscopy demonstrate that mono-SMBGs are internalized by human bone marrow (MSCs) and adipose-derived stem cells (ADSCs) and located within cell vesicles and in the cytoplasm. Particle dissolution inside the cells is observed. Alamar Blue, MTT and Cyquant assays demonstrate that 50 µg mL(-1) of mono-SMBGs did not inhibit significantly MSC or ADSC metabolic activity. However, at higher concentrations (100 and 200 µg mL(-1)) small decrease in metabolic activity and total DNA is observed. Mono-SMBG did not induce ALPase activity, an early marker of osteogenic differentiation, without osteogenic supplements; however, in their presence osteogenic differentiation is achieved. Additionally, large numbers of particles are internalized by the cells but have little effect on cell behavior.

Bioactive glass foam scaffolds are remodelled by osteoclasts and support the formation of mineralized matrix and vascular networks in vitro.

Remodelling of scaffolds and new bone formation is critical for effective bone regeneration. Herein is reported the first demonstration of resorption pits due to osteoclast activity on the surface of sol-gel bioactive glass foam scaffolds. Bioactive glass foam scaffolds are known to have osteogenic potential and suitable pore networks for bone regeneration. Degradation of the scaffolds is known to be initially solution mediated, but for effective bone regeneration, remodelling of the scaffold by osteoclasts and vascularisation of the scaffold is necessary. The culture of C7 macrophages on a bioactive glass scaffold induces the cells to differentiate into (TRAP(+ve) ) osteoclasts. They then form distinctive resorption pits within 3 weeks, while MC3T3-E1 pre-osteoblasts deposit mineralized osteoid on their surfaces in co-culture. The scaffolds are of the 70S30C (70 mol% SiO2 , 30 mol% CaO) composition, with modal pore and interconnect diameters of 373 µm and 172 µm respectively (quantified by X-ray micro-tomography and 3D image analysis). The release of soluble silica and calcium ions from 70S30C scaffolds induces an increase in osteoblast numbers as determined via the MTT assay. Scaffolds also support growth of endothelial cells on their surface and tube formation (characteristic of functional microvasculature) following 4 days in culture. This data supports the hypothesis that 70S30C bioactive glass scaffolds promote the differentiation of the 3 main cell types involved in vascularized bone regeneration.

Spherical bioactive glass particles and their interaction with human mesenchymal stem cells in vitro.

Sub-micron particles of bioactive glass (SMBGs) with composition 85 mol% SiO(2) and 15 mol% CaO were synthesised and characterised. Bioactivity was demonstrated by the formation of calcium apatite following 5 days immersion in simulated body fluid (SBF). The effect of a 24 h exposure of SMBGs (100 µg/ml, 150 µg/ml, 200 µg/ml) to human mesenchymal stem cells (hMSCs) on cell viability, metabolic activity and proliferation were determined using the LIVE/DEAD, MTT, total DNA and LDH assays after 1, 4 and 7 days of culture. None of the SMBG concentrations caused significant cytotoxicity at 1 and 4 days, but the doses of 150 and 200 µg/ml significantly decreased hMSC metabolic activity after 7 days of culture. Cell proliferation decreased as SMBG concentration increased; however none of the SMBGs tested had a significant effect on DNA quantity compared to the control. Confocal microscopy confirmed cellular uptake and localisation of the SMBGs in the hMSC cytoskeleton. Transmission electron microscopy revealed that the SMBGs localised inside the cell cytoplasm and cell endosomes. These findings are important for assessing the toxicity of sub-micron particles that may either be used as injectables for bone regeneration or generated by wear or degradation of bioactive glass scaffolds.