Gavin Jell

The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro.

Bioactive glasses (BG) which contain strontium have the potential to combine the known bone regenerative properties of BG with the anabolic and anti-catabolic effects of strontium cations. Here we created a BG series (SiO(2)-P(2)O(5)-Na(2)O-CaO) in which 0-100% of the calcium was substituted by strontium and tested their effects on osteoblasts and osteoclasts in vitro. We show that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts. They also inhibit osteoclast activity by both reducing tartrate resistant acid phosphatase activity and inhibiting resorption of calcium phosphate films in a dose-dependent manner. Additionally, osteoblasts cultured in contact with BG show increased proliferation and alkaline phosphatase activity with increasing strontium substitution, while osteoclasts adopt typical resorption morphologies. These results suggest that similarly to the osteoporosis drug strontium ranelate, strontium-substituted BG may promote an anabolic effect on osteoblasts and an anti-catabolic effect on osteoclasts. These effects, when combined with the advantages of BG such as controlled ion release and delivery versatility, may make strontium-substituted BG an effective biomaterial choice for a range of bone regeneration therapies.

Biofunctionalization of Biomaterials for Accelerated in Situ Endothelialization: A Review

The higher patency rates of cardiovascular implants, including vascular bypass grafts, stents, and heart valves are related to their ability to inhibit thrombosis, intimal hyperplasia, and calcification. In native tissue, the endothelium plays a major role in inhibiting these processes. Various bioengineering research strategies thereby aspire to induce endothelialization of graft surfaces either prior to implantation or by accelerating in situ graft endothelialization. This article reviews potential bioresponsive molecular components that can be incorporated into (and/or released from) biomaterial surfaces to obtain accelerated in situ endothelialization of vascular grafts. These molecules could promote in situ endothelialization by the mobilization of endothelial progenitor cells (EPC) from the bone marrow, encouraging cell-specific adhesion (endothelial cells (EC) and/or EPC) to the graft and, once attached, by controlling the proliferation and differentiation of these cells. EC and EPC interactions with the extracellular matrix continue to be a principal source of inspiration for material biofunctionalization, and therefore, the latest developments in understanding these interactions will be discussed.

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro‐spectroscopy is a laser‐based technique which enables rapid and non‐invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro‐spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell‐specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G0/G1, S, and G2/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G0/G1 to G2/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G0/G1‐phase cells and G2/M‐phase cells. The non‐invasive analysis of live cell cycle dynamics with Raman micro‐spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels. J. Cell. Biochem. 104: 1427–1438, 2008.

In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy.

Raman micro‐spectroscopy combined with multivariate analysis was employed to monitor real‐time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double‐strand DNA breaks in human type II pneumocyte‐like cells (A549 cell‐line). Raman spectra of A549 cells exposed to 100 µM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over‐expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro‐spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types. J. Cell. Biochem.

In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering.

Raman micro‐spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast‐like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non‐tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non‐tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non‐tumour bone cells. A supervised classification model (LDA) was built and showed high cross‐validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non‐tumour cells, with 96% of the cells being correctly classified either as tumour or non‐tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells.

Carbon nanotube-enhanced polyurethane scaffolds fabricated by thermally induced phase separation

Nanocomposite foams are an attractive prospect in a number of fields including biomedical science, catalysis and filtration. In biomedical engineering, porous nanocomposite scaffolds can potentially mimic aspects of the nanoscale architecture of the extra-cellular matrix, as well as enhance the mechanical properties required for successful weight-bearing implants. Thermoplastic polyurethane–multi-walled carbon nanotubes (CNTs) foams were manufactured by thermally induced phase separation (TIPS). TIPS proved to be a successful manufacturing route to three-dimensional, highly porous polymers containing well-dispersed CNTs. Some CNTs are trapped perpendicular to the pore surface creating a rough, nanotextured surface. The surface character of the nanocomposites became more acidic with increasing loading fraction of oxidised CNTs. However, due to the heterogeneity of the nanocomposite surface, its wetting behaviour was not affected. CNT incorporation significantly improved the compression strength and stiffness of the nanocomposite scaffold. The biological properties of these scaffolds were studied in vitro and revealed that increasing MWNT loading fraction did not cause osteoblast cytotoxicity or detrimental effects on osteoblast differentiation or mineralisation. However, osteoblast production of the potent angiogenic factor VEGF (vascular endothelial growth factor) increased in proportion to CNT loading (after 3 days in culture), revealing the potential of the nanocomposite scaffolds to influence cellular behaviour.

Design and development of nanocomposite scaffolds for auricular reconstruction.

UNLABELLED Auricular reconstruction using sculpted autologous costal cartilage is effective, but complex and time consuming and may incur donor site sequelae and morbidity. Conventional synthetic alternatives are associated with infection and extrusion in up to about 15% of cases. We present a novel POSS-PCU nanocomposite auricular scaffold, which aims to reduce extrusion rates by mimicking the elastic modulus of human ears and by encouraging desirable cellular interactions. The fabrication, physicochemical properties (including nanoscale topography) and cellular interactions of these scaffolds were compared to Medpor®, the current synthetic standard. Our scaffold had a more similar elastic modulus (5.73 ± 0.17MPa) to ear cartilage (5.02 ± 0.17MPa) compared with Medpor®, which was much stiffer (140.9 ± 0.04MPa). POSS-PCU supported fibroblast ingrowth and proliferation; significantly higher collagen production was also produced by cells on the POSS-PCU than those on Medpor®. This porous POSS-PCU nanocomposite scaffold is therefore a promising alternative biomaterial for auricular surgical reconstruction. FROM THE CLINICAL EDITOR In this paper, a novel POSS-PCU nanocomposite auricular scaffold is described to reduce extrusion rates by having a much closer elastic modulus of human ears than the currently available synthetic standard. Enabling desirable cellular interactions may lead to the successful clinical application of these novel scaffolds.

Investigation of Schwann cell behaviour on RGD-functionalised bioabsorbable nanocomposite for peripheral nerve regeneration.

Current commercially available nerve conduits fail to support nerve regeneration gaps larger than 30 mm in length due to the simple intra-luminal design of these conduits which are unable to biomimic the native neural environment. There is, therefore, a major clinical demand for new smart biomaterials, which can stimulate neuronal cell proliferation and migration, and facilitate nerve regeneration across these critical sized defects. In this study, we aimed to investigate Schwann cell (SC) behaviour seeded on the bioabsorbable version of the nanocomposite material, POSS modified poly (caprolactone) urea urethane (PCL), functionalised with arginine-glycine-aspartic acid (RGD) peptide. Successful synthesis of RGD peptide as well as the chemical structure of POSS-PCL nanocomposite film was investigated by Fourier transform infrared spectroscopy. Cell viability assay and morphological assessment were performed to investigate the cytocompatibility of the fabricated constructs. Successful immobilisation of RGD peptide onto the nanocomposite surface was confirmed by water contact angle, Brilliant Blue (BB) staining and thin layer chromatography. Both POSS-PCL and RGD-POSS-PCL nanocomposite scaffolds supported SC attachment, proliferation and morphological differentiation, important aspects for peripheral nerve regeneration. However, a significant increase in SC process length and morphological differentiation towards maturation was observed on the cells grown on RGD-POSS-PCL film. RGD-POSS-PCL nanocomposite demonstrated a significant improvement in SCs spreading and its integrin-dependent process outgrowth (P<0.05). Conduits made by POSS-nanocomposite may be suitable for the next generation of commercially available conduit required to meet current clinical demand in peripheral nerve regeneration and repair as they are currently undergoing in vivo preclinical study.

Indirect Cytotoxicity Evaluation of Silver Doped Bioglass Ag-S70C30 on Human Primary Keratinocytes

Bioactive gel-glasses, such as the silver-doped Ag-S70C30 glass, can be used to modify the inflammatory response in a local body compartment such as in acne lesions and in nonhealing dermal wounds. In this study, the cytotoxicity of soluble silver, calcium and silica ions on human epidermal keratinocytes was investigated by measurements of mitochondrial activity (MTT assay) and neutral red dye uptake (NR assay). Ag-S70C30 extracts were prepared by soaking glass powder in complete culture medium at concentrations of 1 mg/ml and 2 mg/ml (mg of glass powder per ml of culture medium). Silver concentrations for both concentrations of approximately 1 ppm were detected by inductive coupled plasma analysis (ICP). No negative effect on the cell viability was measured for an initial gel-glass concentration of 1 mg/ml and for the two shortest extraction times at a concentration of 2 mg/ml. Based on the results from MTT/ NR assays, a pH rise of approximately one unit had no negative effect on the NHEK-A cell viability. This preliminary study on keratinocyte viability merits future investigations on silver bioglass as a novel antimicrobial wound healing agent.

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.