Zinc and strontium based phosphate glass beads: a novel material for bone tissue engineering.

Degradable phosphate-based glasses that contain strontium, zinc and calcium were investigated to examine their function as an osteoconductive material. Glass beads of the general formula of (P2O5)-(Na2O)-(TiO2)-(CaO)-(SrO) or (ZnO) were prepared by a melt quench technique followed by milling and spheroidisation. After performing x-ray diffraction on all the samples for glass structure evaluation, glass bead size distribution was initially measured by a scanning electron microscope (SEM). Then, some of these samples were immersed in deionised water to evaluate both the surface changes and measure the ion release rate, whereas other samples of glass beads were incubated in culture media to determine pH changes. Furthermore, human osteoblast-like osteosarcoma cells MG63 and human mesenchymal stem cells were seeded on the glass beads to determine their cytocompatibility via applying CCK assay, ALP assay and Ca assay. SEM images and fluorescence images of confocal microscopy were performed for the cellular studies. While mass degradation and ion release results displayed a significant increase with zinc and strontium incorporation within time, pH results showed an initial increase in pH followed by a decrease. Cellular studies emphasised that all formulations enhanced cellular proliferation. Phosphate glass beads with zinc content 5 mol% and strontium content of 17.5 mol%, (ZnO5) and (SrO17.5) respectively displayed more promising results although they were insignificantly different from that of control (p > 0.05). This may suggest their applicability in hard tissue engineering.

Chondrogenic potential of blood-acquired mesenchymal progenitor cells.

INTRODUCTION: The associated morbidity from the acquisition of mesenchymal stem cells (MSC) from the bone marrow has led to the investigation of alternative stem cell sources. We propose that such cells may be isolated from non-mobilised blood and demonstrate their differentiation into a chondrocytic lineage. This safe and abundant source of cells may be useful for tissue engineering cartilage. METHOD: Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults and cultured in RPMI medium supplemented with serum. The non-adherent and adherent cells were analysed for cell surface marker expression of CD14, CD34, CD133, CD105 and CD45 by flow cytometry. Adherent cells were also cultured on glass slides in chondrogenic media and analysed for the expression of collagen I and II on day 14 of culture. RESULTS: The adherent cells were fibroblastic in morphology and were confluent on day 14. The non-adherent and adherent cell populations were shown to have distinct profiles by flow cytometry. The adherent cells were positive for CD105 and CD14 and also expressed collagen I and II precursors when cultured in chondrogenic media. CONCLUSION: Blood-acquired mesenchymal progenitor cells (BMPCs) can be isolated from non-mobilised blood. These unique cells are CD105(+) and CD14(+) and have chondrogenic differentiation capacity. BMPC may provide a potential source of MPC for tissue engineering applications.

Changes in bone morphogenetic protein receptor-IB localisation regulate osteogenic responses of human bone cells to bone morphogenetic protein-2.

Cell responses to bone morphogenetic proteins (BMP) depend on the expression and surface localisation of transmembrane receptors BMPR-IA, -IB and -II. The present study shows that all three antigens are readily detected in human bone cells. However, only BMPR-II was found primarily at the plasma membrane, whereas BMPR-IA was expressed equally in the cytoplasm and at the cell surface. Notably, BMPR-IB was mainly intracellular, where it was associated with a number of cytoplasmic structures and possibly the nucleus. Treatment with transforming growth factor beta1 (TGF-beta1) caused rapid translocation of BMPR-IB to the cell surface, mediated via the p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. The TGF-beta1-induced increase in surface BMPR-IB resulted in significantly elevated BMP-2 binding and Smad1/5/8 phosphorylation, although the receptor was subsequently internalised and the functional response to BMP-2 consequently down-regulated. The results show, for the first time, that BMPR-IB is localised primarily in intracellular compartments in bone cells and that TGF-beta1 induces rapid surface translocation from the cytoplasm to the cell surface, resulting in increased sensitivity of the cells to BMP-2.

Novel adhesin from Pasteurella multocida that binds to the integrin-binding fibronectin FnIII9-10 repeats.

Phage display screening with fragmented genomic DNA from the animal pathogen Pasteurella multocida has identified a gene encoding a putative fibronectin binding protein (19). Homologues of this gene (PM1665) are found in all other sequenced members of the Pasteurellaceae. Gene PM1665 has been cloned, and the protein has been expressed. Recombinant PM1665 protein binds to both soluble and immobilized fibronectin and is unique in that it interacts with the integrin-binding fibronectin type III (FnIII) repeats FnIII(9-10) and not, as is the case for almost all other fibronectin adhesins, to the N-terminal type I repeats. Surface plasmon resonance analysis revealed a complex binding mechanism with a K(D) (equilibrium dissociation constant) of 150 nM +/- 70 nM. Bioinformatics analysis suggests that the PM1665 protein contains two helix-hairpin-helix (HhH) motifs, and truncation mutation studies have identified the binding site in the protein as a combination of these two HhH motifs in conjunction with a conserved amino acid motif, VNINTA. We have shown that the PM1665 protein is on the cell surface and that binding of P. multocida to fibronectin is almost completely inhibited by anti-PM1665 antiserum. These results support the hypothesis that the PM1665 protein is a member of a new family of fibronectin binding adhesins that are important in the adhesion of P. multocida to fibronectin.

Initial responses of human osteoblasts to sol-gel modified titanium with hydroxyapatite and titania composition.

Sol-gel thin films of hydroxyapatite (HA) and titania (TiO(2)) have received a great deal of attention in the area of bioactive surface modification of titanium (Ti) implants. Sol-gel coatings were developed on Ti substrates of pure HA and TiO(2) and two composite forms, HA+10% TiO(2) and HA+20% TiO(2), and the biological properties of the coatings were evaluated. All the coating layers exhibited thin and homogeneous structures and phase-pure compositions (either HA or TiO(2)). Primary human osteoblast cells showed good attachment, spreading and proliferation on all the sol-gel coated surfaces, with enhanced cell numbers on all the coated surfaces relative to uncoated Ti control at day 1, as observed by MTT assay and scanning electron microscopy. Cell attachment rates were also enhanced on the pure HA coating relative to control Ti. The pure HA and HA+10% TiO(2) composite coating furthermore enhanced proliferation of osteoblasts at 4 days. Moreover, the gene expression level of several osteogenic markers including bone sialoprotein and osteopontin, as measured by RT-PCR at 24h, was shown to vary according to coating composition. These findings suggest that human primary bone cells show marked and rapid early functional changes in response to HA and TiO(2) sol-gel coatings on Ti.

Surface characterisation of various bone cements prepared with functionalised methacrylates/bioactive ceramics in relation to HOB behaviour.

This study reports the relationship between the biocompatibility and surface properties of experimental bone cements. The effect of hydroxyapatite (HA) or alpha-tri-calcium phosphate (alpha-TCP) incorporated into bone cements prepared with methyl methacrylate as base monomer and either methacrylic acid or diethyl amino ethyl methacrylate (DEAEMA) as comonomers was investigated. The in vitro biocompatibility of these composite cements was assessed in terms of the interaction of primary human osteoblasts grown on the materials over a period of 5 days and compared with a control surface. These results were related to the surface properties investigated through a number of techniques, namely Fourier transform infrared, contact angle measurements, X-ray photoelectron spectroscopy and energy dispersive analysis of X-rays. Complementary techniques of thermal analysis and ion chromatography were also performed. Biocompatibility results showed that the addition of alpha-TCP improves biocompatibility regardless of comonomer type. This is in contrast to HA-based cements where cell proliferation was significantly lower. Surface characterisations showed that structural integrity of the materials was maintained in the presence of the acid and base comonomers, and water contact angles were reduced particularly in DEAEMA containing materials. Furthermore, ion chromatography confirmed higher Ca2+ and PO4(3-) ion release by both types of ceramics, particularly for those containing DEAEMA. In conclusion, the incorporation of acidic and basic comonomers to either HA or alpha-TCP ceramics containing bone cements can have differential effects upon the attachment and proliferation of bone cells in vitro. Moreover, those cements consisting of alpha-TCP and containing DEAEMA comonomer indicated the most favourable biocompatibility.

An in vitro study into the corrosion of intra-oral magnets in the presence of dental amalgam.

The aim of this investigation was to study the corrosion behaviour and products of uncoated neodymium-iron-boron magnets in the presence of dental amalgam. Microcosm plaques were grown on discs of neodymium-iron-boron magnets or amalgam in a constant depth film fermentor. The biofilms were supplied with artificial saliva and growth was determined by viable counting. The results showed that the neodymium-iron-boron magnets corroded with an average daily weight loss of 0.115 +/- 0.032 per cent. However, when the magnets were in close proximity to the amalgam the amount of corrosion was reduced to a daily loss of 0.066 +/- 0.023 per cent. The highest loss of constituent elements from the corrosion products of the magnets was observed for iron. The composition of the microcosm plaques altered markedly between the two materials with less streptococci and more Veillonella spp. present in the biofilms grown on magnets in the presence of amalgam. The corrosion of neodymium-iron-boron magnets is limited and in the presence of amalgam is reduced further. This suggests that amalgam present in the mouth will not cause an increased clinical risk in terms of biocompatibility with neodymium-iron-boron magnets.