O. Tsigkou - Academia.edu (original) (raw)
Papers by O. Tsigkou
Journal of Vascular and Interventional Radiology, 2013
Purpose: To assess the safety and feasibility of the targeted delivery of the antiangiogenic drug... more Purpose: To assess the safety and feasibility of the targeted delivery of the antiangiogenic drug sorafenib to the liver using transarterial chemoembolization methodology as a novel approach to hepatocellular carcinoma (HCC) therapy. Materials and Methods: Seven healthy New Zealand white rabbits were used in the study. After placement of a catheter in the common hepatic artery, six rabbits were treated with chemoembolization of sorafenib in iodized oil (Lipiodol) (sorafenib dose 0.1 mg/kg), and one rabbit received Lipiodol only. Liquid chromatography tandem mass spectrometry was used to measure the concentration of sorafenib in the peripheral blood and liver tissue 24 hours and 72 hours after treatment. Histochemical staining of the liver sections and biochemical measurements were performed. Results: The administration of sorafenib in Lipiodol emulsions by transarterial chemoembolization resulted in sorafenib concentrations of 794 ng/g Ϯ 240 and 64 ng/g Ϯ 15 in the liver tissue 24 hours and 72 hours after treatment. The average liverto-serum ratios 24 hours and 72 hours after treatment were approximately 14 and 22. The histochemical staining of the liver tissue sections and aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase and total bilirubin concentrations indicated no significant liver damage. Conclusions: Transarterial chemoembolization with sorafenib in Lipiodol is an effective methodology for the localized delivery of this drug to the liver and has possible practical implications in therapeutic interventions for the treatment of hepatocellular carcinoma. ABBREVIATIONS AcCN = acetonitrile, ALT = alanine aminotransferase, AST = aspartate aminotransferase, GGT = γ-glutamyltransferase, HCC = hepatocellular carcinoma, LC-ESI-MS/MS = liquid chromatography electrospray ionization mass spectrometry, LC-MS/MS = liquid chromatography tandem mass spectrometry
Tissue engineering. Part C, Methods, Jan 12, 2015
Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are t... more Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation and robust 3D post image processing are essential for evaluation but have not been applied previously because of technical difficulties. Here we report novel video rate confocal microscopy and 3D post image processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (DL-lactide-co-glycolide) (PLGA) scaffold. Video rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the micro circulation of the blood cells. Post image processing was applied to perform 3D image reconstructio...
Proceedings of the National Academy of Sciences, 2010
Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone graft... more Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4–7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium des...
European Cells & Materials, 2009
Experimental Eye Research, 2012
Culturing corneal keratocytes is difficult because keratocytes growing in a monolayer rapidly los... more Culturing corneal keratocytes is difficult because keratocytes growing in a monolayer rapidly lose their stellate morphology and cease to express keratocyte markers such as keratocan, lumican and aldehyde dehydrogenase 1 family, member A1 (ALDH1A1). Conversely, mesenchymal stem cells (MSCs) can be easily expanded in cell culture, and they have a variety of differentiation pathways. We studied the feasibility of using MSCs as a source for corneal tissue engineering. Based on the observation that keratocytes have MSC-like properties, similar to bone marrow-derived MSCs (BM-MSCs), we hypothesized that MSCs would differentiate into corneal keratocyte-like cells in keratocyte-conditioned medium (KCM). We measured changes in the expression of keratocyte markers through quantitative real-time polymerase chain reaction (qRT-PCR) and found that human MSC's cultured in KCM expressed both keratocan and ALDH1A1. Western blot analysis demonstrated that human MSCs cultured in KCM steadily increased their expression of lumican and ALDH1A1, while they lost expression of a-smooth muscle actin (a-SMA). Immunocytochemistry indicated that human MSCs grown in KCM acquired characteristics similar to those of keratocytes. These results suggest that KCM can direct human MSCs to differentiate into keratocyte-like cells.
Tissue engineering. Part A, 2015
Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, p... more 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.
Materials Today Advances
Triple-negative breast cancers are extremely aggressive with limited treatment options because of... more Triple-negative breast cancers are extremely aggressive with limited treatment options because of the reduced response of the cancerous cells to hormonal therapy. Here, monodispersed zinc-containing mesoporous silica nanoparticles (MSNPs-Zn) were produced as a tuneable biodegradable platform for delivery of therapeutic zinc ions into cells. We demonstrate that the nanoparticles were internalized by cells, and a therapeutic dose window was identified in which the MSNPs-Zn were toxic to breast cancer cells but not to healthy epithelial (MCF-10a) cells or to murine macrophages. A significant reduction in the viability of triple negative MDA-MB-231 and MCF-7 (ERþ) breast cancer cells was seen following 24 h exposure to MSNPs-Zn. The more aggressive MDA-MB-231 cells, with higher metastatic potential, were more sensitive to MSNPs-Zn than the MCF-7 cells. MSNPs-Zn underwent biodegradation inside the cells, becoming hollow structures, as imaged by high-resolution transmission electron microscopy. The mesoporous silica nanoparticles provide a biodegradable vehicle for therapeutic ion release inside cells.
Advanced Healthcare Materials, 2014
Spherical monodispersed bioactive particles are potential candidates for nanocomposite synthesis ... more 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.
ABSTRACT Introduction: Bioactive glasses (BG) are promising for hard tissue regeneration because ... more ABSTRACT Introduction: Bioactive glasses (BG) are promising for hard tissue regeneration because of their rapid bone bonding, controlled biodegradability and their ability to stimulate osteogenesis [1]. BG nanoparticles have the potential to be injected directly into the defect site to allow healing and regeneration of bone tissue. Also, regardless of the great potential of BG as porous scaffolds for bone regeneration concerns arise on their long term fate in the body as small particles may be released after implantation, which could lead to undesirable reactions to surrounding cells, hence investigations on such nanoparticles is crucial. Nanostructured materials possess unique properties that are strongly dependent on size, chemistry and shape, which are of particular interest considering many biological processes occur at nanoscale. As mesenchymal stem cells (MSCs) are precursors to osteoblasts, the effect of nanoparticles on their behaviour is critical. In this study 80S20C (80 mol% SiO 2 and 20 mol% CaO) BG nanoparticles, have been synthesised and characterized for the first time. The BG nanoparticle's uptake and distribution inside MSCs using confocal microscopy and transmission electron microscopy (TEM) was also assessed. The effect of the BG nanoparticles on cell viability, metabolic activity and proliferation as a result of particle uptake was also determined. Materials and Methods: The Stöber process [2] was applied to produce sol-gel derived BG nanoparticles. To follow the internalisation and intracellular distribution of the BG nanoparticles inside MSCs (Lonza, UK) in 3D, cells were exposed to BG nanoparticles at a concentration of 100μg/ml in cell medium (Dublecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin (all from Invitrogen, UK) for 24 h. For confocal microscopy the actin cytoskeleton was stained with Alexa Fluor conjugated phaloidin (molecular probes, UK). TEM was also used to monitor the uptake and distribution of the nanoparticles (100μg/ml) inside MSCs after 24 h exposure. Cells were fixed, osmicated and the samples were embedded in resin and sectioned. The effect of the nanoparticles on cell viability and proliferation was determined by exposing MSCs to three different BG nanoparticle concentrations: 100, 150 and 200 μg/ml in cell medium (DMEM) for 24h and their response monitored over of the period of 1, 4 and 7 days using LIVE/DEAD (Molecular Probes, UK), MTT 3-(4,5-Dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide (Sigma, UK), total DNA using Hoechst (Sigma, UK) (and Lactate dehydrogenase (LDH) Cytotox-one TM (Promega, UK).
Tissue engineering. Part C, Methods, Jan 12, 2015
Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are t... more Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation and robust 3D post image processing are essential for evaluation but have not been applied previously because of technical difficulties. Here we report novel video rate confocal microscopy and 3D post image processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (DL-lactide-co-glycolide) (PLGA) scaffold. Video rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the micro circulation of the blood cells. Post image processing was applied to perform 3D image reconstructio...
Soft Matter, 2012
It is well known that bone has excellent mechanical properties through its hierarchical structure... more It is well known that bone has excellent mechanical properties through its hierarchical structure and that design of new materials should take inspiration from its structure, especially those that will be used as synthetic bone grafts. However it has not yet been possible to mimic the complex structure. Here we present new organic–inorganic hybrid scaffolds produced by a sol–gel foaming process with the aim of producing materials that mimic the trabecular structure of bone and take inspiration from the nanocomposite ...
Journal of the European Ceramic Society, 2012
ABSTRACT The fabrication of a new composite glass–ceramic with potential application in dental re... more ABSTRACT The fabrication of a new composite glass–ceramic with potential application in dental restoration was investigated. The developed material aims to modify the surface of dental ceramics creating bioactive surfaces able to improve material–cell interaction enhancing the bonding of the marginal gap between restoration and tooth. The application of the sol–gel method led to a microporous homogeneous glass–ceramic which can be applied as coating on commercial dental ceramic substrates. The microstructural, thermal, mechanical and biological properties of the fabricated coatings were studied and compared to the respective results of a previously investigated glass–ceramic composite. The material–cell interaction on these two sol–gel dental composites was studied in detail. The attachment and proliferation of both periodontal ligament and gingival fibroblast cells confirmed the bioactive behavior of the new materials and their ability to be potentially applied in dental restorations for soft tissue regeneration and sealing of the marginal gap.
Journal of Biomedical Materials Research Part A, 2007
This study investigates the cellular response of fetal osteoblasts to bioactive resorbable compos... more This study investigates the cellular response of fetal osteoblasts to bioactive resorbable composite films consisting of a poly-D,L-lactide (PDLLA) matrix and bioactive glass 45S5 Bioglass 1 (BG) particles at three different concentrations (0% (PDLLA), 5% (P/BG5), and 40% (P/ BG40)). Using scanning electron microscopy (SEM) we observed that cells were less spread and elongated on PDLLA and P/BG5, whereas cells on P/BG40 were elongated but with multiple protrusions spreading over the BG particles. Vinculin immunostaining revealed similar distribution of focal adhesion contacts on all cells independent of substratum, indicating that all materials permitted cell adhesion. However, when differentiation and maturation of fetal osteoblasts was examined, incorporation of 45S5 BG within the PDLLA matrix was found to significantly (p < 0.05) enhance alkaline phosphatase enzymatic activity and osteocalcin protein synthesis compared to tissue cul-ture polystyrene controls and PDLLA alone. Alizarin red staining indicated extracellular matrix mineralization on both P/BG5 and P/BG40, with significantly more bone nodules formed than on PDLLA. Real time RT-PCR revealed that expression of bone sialoprotein was also affected by the BG containing films compared to controls, whereas expression of Collagen Type I was not influenced. By performing these investigations in the absence of osteogenic factors it appears that the incorporation of BG stimulates osteoblast differentiation and mineralization of the extracellular matrix, demonstrating the osteoinductive capacity of the composite.
Journal of Biomedical Materials Research Part A, 2008
Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect re... more Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect realtime biochemical changes in foetal osteoblasts (FOBs) following exposure to 45S5 Bioglass 1 (BG)-conditioned media. Bio-Raman spectroscopy, combined with multivariate statistical analysis techniques (principal component analysis and least squares analysis), was able to noninvasively identify biochemical differences in FOBs cultured for different time periods and between FOBs exposed/or not to BG-conditioned media. Gene and protein expression studies were also performed for known markers of osteoblastic differentiation, namely, alkaline phosphatase, bone sialoprotein, and collagen type I. Quantitative RT-PCR con-firmed upregulation of genes associated with osteoblast differentiation after exposure to BG-conditioned media. These results suggest that Raman spectroscopy can noninvasively detect biochemical changes in FOBs associated with differentiation. This technique could have important applications in the field of regenerative medicine by enabling rapid characterization of cell or organoid behavior on novel bioactive scaffolds without damage to either cell or biomaterial.
Biomaterials, 2009
Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previou... more Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass Ò conditioned medium with two different Si concentrations (15 mg/ml (BGCM/15) and 20 mg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass Ò conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/ 15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of b-glycerophosphate, L-ascorbate-2phosphate or dexamethasone (commonly used osteogenic supplements).
Biomaterials, 2011
Sub-micron particles of bioactive glass (SMBGs) with composition 85 mol% SiO 2 and 15 mol% CaO we... more 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 mg/ml, 150 mg/ml, 200 mg/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 mg/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.
Journal of Vascular and Interventional Radiology, 2013
Purpose: To assess the safety and feasibility of the targeted delivery of the antiangiogenic drug... more Purpose: To assess the safety and feasibility of the targeted delivery of the antiangiogenic drug sorafenib to the liver using transarterial chemoembolization methodology as a novel approach to hepatocellular carcinoma (HCC) therapy. Materials and Methods: Seven healthy New Zealand white rabbits were used in the study. After placement of a catheter in the common hepatic artery, six rabbits were treated with chemoembolization of sorafenib in iodized oil (Lipiodol) (sorafenib dose 0.1 mg/kg), and one rabbit received Lipiodol only. Liquid chromatography tandem mass spectrometry was used to measure the concentration of sorafenib in the peripheral blood and liver tissue 24 hours and 72 hours after treatment. Histochemical staining of the liver sections and biochemical measurements were performed. Results: The administration of sorafenib in Lipiodol emulsions by transarterial chemoembolization resulted in sorafenib concentrations of 794 ng/g Ϯ 240 and 64 ng/g Ϯ 15 in the liver tissue 24 hours and 72 hours after treatment. The average liverto-serum ratios 24 hours and 72 hours after treatment were approximately 14 and 22. The histochemical staining of the liver tissue sections and aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase and total bilirubin concentrations indicated no significant liver damage. Conclusions: Transarterial chemoembolization with sorafenib in Lipiodol is an effective methodology for the localized delivery of this drug to the liver and has possible practical implications in therapeutic interventions for the treatment of hepatocellular carcinoma. ABBREVIATIONS AcCN = acetonitrile, ALT = alanine aminotransferase, AST = aspartate aminotransferase, GGT = γ-glutamyltransferase, HCC = hepatocellular carcinoma, LC-ESI-MS/MS = liquid chromatography electrospray ionization mass spectrometry, LC-MS/MS = liquid chromatography tandem mass spectrometry
Tissue engineering. Part C, Methods, Jan 12, 2015
Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are t... more Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation and robust 3D post image processing are essential for evaluation but have not been applied previously because of technical difficulties. Here we report novel video rate confocal microscopy and 3D post image processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (DL-lactide-co-glycolide) (PLGA) scaffold. Video rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the micro circulation of the blood cells. Post image processing was applied to perform 3D image reconstructio...
Proceedings of the National Academy of Sciences, 2010
Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone graft... more Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4–7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium des...
European Cells & Materials, 2009
Experimental Eye Research, 2012
Culturing corneal keratocytes is difficult because keratocytes growing in a monolayer rapidly los... more Culturing corneal keratocytes is difficult because keratocytes growing in a monolayer rapidly lose their stellate morphology and cease to express keratocyte markers such as keratocan, lumican and aldehyde dehydrogenase 1 family, member A1 (ALDH1A1). Conversely, mesenchymal stem cells (MSCs) can be easily expanded in cell culture, and they have a variety of differentiation pathways. We studied the feasibility of using MSCs as a source for corneal tissue engineering. Based on the observation that keratocytes have MSC-like properties, similar to bone marrow-derived MSCs (BM-MSCs), we hypothesized that MSCs would differentiate into corneal keratocyte-like cells in keratocyte-conditioned medium (KCM). We measured changes in the expression of keratocyte markers through quantitative real-time polymerase chain reaction (qRT-PCR) and found that human MSC's cultured in KCM expressed both keratocan and ALDH1A1. Western blot analysis demonstrated that human MSCs cultured in KCM steadily increased their expression of lumican and ALDH1A1, while they lost expression of a-smooth muscle actin (a-SMA). Immunocytochemistry indicated that human MSCs grown in KCM acquired characteristics similar to those of keratocytes. These results suggest that KCM can direct human MSCs to differentiate into keratocyte-like cells.
Tissue engineering. Part A, 2015
Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, p... more 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.
Materials Today Advances
Triple-negative breast cancers are extremely aggressive with limited treatment options because of... more Triple-negative breast cancers are extremely aggressive with limited treatment options because of the reduced response of the cancerous cells to hormonal therapy. Here, monodispersed zinc-containing mesoporous silica nanoparticles (MSNPs-Zn) were produced as a tuneable biodegradable platform for delivery of therapeutic zinc ions into cells. We demonstrate that the nanoparticles were internalized by cells, and a therapeutic dose window was identified in which the MSNPs-Zn were toxic to breast cancer cells but not to healthy epithelial (MCF-10a) cells or to murine macrophages. A significant reduction in the viability of triple negative MDA-MB-231 and MCF-7 (ERþ) breast cancer cells was seen following 24 h exposure to MSNPs-Zn. The more aggressive MDA-MB-231 cells, with higher metastatic potential, were more sensitive to MSNPs-Zn than the MCF-7 cells. MSNPs-Zn underwent biodegradation inside the cells, becoming hollow structures, as imaged by high-resolution transmission electron microscopy. The mesoporous silica nanoparticles provide a biodegradable vehicle for therapeutic ion release inside cells.
Advanced Healthcare Materials, 2014
Spherical monodispersed bioactive particles are potential candidates for nanocomposite synthesis ... more 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.
ABSTRACT Introduction: Bioactive glasses (BG) are promising for hard tissue regeneration because ... more ABSTRACT Introduction: Bioactive glasses (BG) are promising for hard tissue regeneration because of their rapid bone bonding, controlled biodegradability and their ability to stimulate osteogenesis [1]. BG nanoparticles have the potential to be injected directly into the defect site to allow healing and regeneration of bone tissue. Also, regardless of the great potential of BG as porous scaffolds for bone regeneration concerns arise on their long term fate in the body as small particles may be released after implantation, which could lead to undesirable reactions to surrounding cells, hence investigations on such nanoparticles is crucial. Nanostructured materials possess unique properties that are strongly dependent on size, chemistry and shape, which are of particular interest considering many biological processes occur at nanoscale. As mesenchymal stem cells (MSCs) are precursors to osteoblasts, the effect of nanoparticles on their behaviour is critical. In this study 80S20C (80 mol% SiO 2 and 20 mol% CaO) BG nanoparticles, have been synthesised and characterized for the first time. The BG nanoparticle's uptake and distribution inside MSCs using confocal microscopy and transmission electron microscopy (TEM) was also assessed. The effect of the BG nanoparticles on cell viability, metabolic activity and proliferation as a result of particle uptake was also determined. Materials and Methods: The Stöber process [2] was applied to produce sol-gel derived BG nanoparticles. To follow the internalisation and intracellular distribution of the BG nanoparticles inside MSCs (Lonza, UK) in 3D, cells were exposed to BG nanoparticles at a concentration of 100μg/ml in cell medium (Dublecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin (all from Invitrogen, UK) for 24 h. For confocal microscopy the actin cytoskeleton was stained with Alexa Fluor conjugated phaloidin (molecular probes, UK). TEM was also used to monitor the uptake and distribution of the nanoparticles (100μg/ml) inside MSCs after 24 h exposure. Cells were fixed, osmicated and the samples were embedded in resin and sectioned. The effect of the nanoparticles on cell viability and proliferation was determined by exposing MSCs to three different BG nanoparticle concentrations: 100, 150 and 200 μg/ml in cell medium (DMEM) for 24h and their response monitored over of the period of 1, 4 and 7 days using LIVE/DEAD (Molecular Probes, UK), MTT 3-(4,5-Dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide (Sigma, UK), total DNA using Hoechst (Sigma, UK) (and Lactate dehydrogenase (LDH) Cytotox-one TM (Promega, UK).
Tissue engineering. Part C, Methods, Jan 12, 2015
Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are t... more Vascularization is a key challenge in tissue engineering. 3D structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation and robust 3D post image processing are essential for evaluation but have not been applied previously because of technical difficulties. Here we report novel video rate confocal microscopy and 3D post image processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (DL-lactide-co-glycolide) (PLGA) scaffold. Video rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the micro circulation of the blood cells. Post image processing was applied to perform 3D image reconstructio...
Soft Matter, 2012
It is well known that bone has excellent mechanical properties through its hierarchical structure... more It is well known that bone has excellent mechanical properties through its hierarchical structure and that design of new materials should take inspiration from its structure, especially those that will be used as synthetic bone grafts. However it has not yet been possible to mimic the complex structure. Here we present new organic–inorganic hybrid scaffolds produced by a sol–gel foaming process with the aim of producing materials that mimic the trabecular structure of bone and take inspiration from the nanocomposite ...
Journal of the European Ceramic Society, 2012
ABSTRACT The fabrication of a new composite glass–ceramic with potential application in dental re... more ABSTRACT The fabrication of a new composite glass–ceramic with potential application in dental restoration was investigated. The developed material aims to modify the surface of dental ceramics creating bioactive surfaces able to improve material–cell interaction enhancing the bonding of the marginal gap between restoration and tooth. The application of the sol–gel method led to a microporous homogeneous glass–ceramic which can be applied as coating on commercial dental ceramic substrates. The microstructural, thermal, mechanical and biological properties of the fabricated coatings were studied and compared to the respective results of a previously investigated glass–ceramic composite. The material–cell interaction on these two sol–gel dental composites was studied in detail. The attachment and proliferation of both periodontal ligament and gingival fibroblast cells confirmed the bioactive behavior of the new materials and their ability to be potentially applied in dental restorations for soft tissue regeneration and sealing of the marginal gap.
Journal of Biomedical Materials Research Part A, 2007
This study investigates the cellular response of fetal osteoblasts to bioactive resorbable compos... more This study investigates the cellular response of fetal osteoblasts to bioactive resorbable composite films consisting of a poly-D,L-lactide (PDLLA) matrix and bioactive glass 45S5 Bioglass 1 (BG) particles at three different concentrations (0% (PDLLA), 5% (P/BG5), and 40% (P/ BG40)). Using scanning electron microscopy (SEM) we observed that cells were less spread and elongated on PDLLA and P/BG5, whereas cells on P/BG40 were elongated but with multiple protrusions spreading over the BG particles. Vinculin immunostaining revealed similar distribution of focal adhesion contacts on all cells independent of substratum, indicating that all materials permitted cell adhesion. However, when differentiation and maturation of fetal osteoblasts was examined, incorporation of 45S5 BG within the PDLLA matrix was found to significantly (p < 0.05) enhance alkaline phosphatase enzymatic activity and osteocalcin protein synthesis compared to tissue cul-ture polystyrene controls and PDLLA alone. Alizarin red staining indicated extracellular matrix mineralization on both P/BG5 and P/BG40, with significantly more bone nodules formed than on PDLLA. Real time RT-PCR revealed that expression of bone sialoprotein was also affected by the BG containing films compared to controls, whereas expression of Collagen Type I was not influenced. By performing these investigations in the absence of osteogenic factors it appears that the incorporation of BG stimulates osteoblast differentiation and mineralization of the extracellular matrix, demonstrating the osteoinductive capacity of the composite.
Journal of Biomedical Materials Research Part A, 2008
Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect re... more Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect realtime biochemical changes in foetal osteoblasts (FOBs) following exposure to 45S5 Bioglass 1 (BG)-conditioned media. Bio-Raman spectroscopy, combined with multivariate statistical analysis techniques (principal component analysis and least squares analysis), was able to noninvasively identify biochemical differences in FOBs cultured for different time periods and between FOBs exposed/or not to BG-conditioned media. Gene and protein expression studies were also performed for known markers of osteoblastic differentiation, namely, alkaline phosphatase, bone sialoprotein, and collagen type I. Quantitative RT-PCR con-firmed upregulation of genes associated with osteoblast differentiation after exposure to BG-conditioned media. These results suggest that Raman spectroscopy can noninvasively detect biochemical changes in FOBs associated with differentiation. This technique could have important applications in the field of regenerative medicine by enabling rapid characterization of cell or organoid behavior on novel bioactive scaffolds without damage to either cell or biomaterial.
Biomaterials, 2009
Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previou... more Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass Ò conditioned medium with two different Si concentrations (15 mg/ml (BGCM/15) and 20 mg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass Ò conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/ 15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of b-glycerophosphate, L-ascorbate-2phosphate or dexamethasone (commonly used osteogenic supplements).
Biomaterials, 2011
Sub-micron particles of bioactive glass (SMBGs) with composition 85 mol% SiO 2 and 15 mol% CaO we... more 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 mg/ml, 150 mg/ml, 200 mg/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 mg/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.