Ultrafine-grained porous titanium and porous titanium/magnesium composites fabricated by space holder-enabled severe plastic deformation.

Compaction of powders by equal channel angular pressing (ECAP) using a novel space holder method was employed to fabricate metallic scaffolds with tuneable porosity. Porous Ti and Ti/Mg composites with 60% and 50% percolating porosity were fabricated using powder blends with two kinds of sacrificial space holders. The high compressive strength and good ductility of porous Ti and porous Ti/Mg obtained in this way are believed to be associated with the ultrafine grain structure of the pore walls. To understand this, a detailed electron microscopy investigation was employed to analyse the interface between Ti/Ti and Ti/Mg particles, the grain structures in Ti particles and the topography of pore surfaces. It was found that using the proposed compaction method, high quality bonding between particles was obtained. Comparing with other powder metallurgy methods to fabricate Ti with an open porous structure, where thermal energy supplied by a laser beam or high temperature sintering is essential, the ECAP process conducted at a relatively low temperature of 400°C was shown to produce unique properties.

Biomaterials for spinal cord regeneration: outgrowth of presumptive neuronal precursors on electrospun poly(epsilon)-caprolactone scaffolds microlayered with alternating polyelectrolytes.

The aim of this study was to assess the feasibility of electrospun poly(epsilon)-caprolactone (PCL) scaffolds treated with alternating paly-electrolytes as a controllable three-dimensional adhesive substrate for neuronal progenitors. Unmodified PCL surfaces were generally not supportive of mouse embryonic stem cell (mESC) colony adhesion. However, scaffolds surfaced using layer-by-layer (LbL) deposition of heparin/poly-L-lysine encouraged better local adhesion of mESC colonies, and networking of monolayers containing nestin-positive presumptive neurons, similar to laminin coated controls, as observed by immuno-fluorescence microscopy. Confocal microscopy further revealed depth-wise penetration of mESC nestin-positive cell populations, and orientation along grass topographical features in the LbL scaffolds. LbL deposition therefore appears to provide a satisfactory adhesive substrate for contact and mechanical guidance of neuronal outgrowth in three-dimensions.

Improved oxygen diffusion and mechanical aggregation of tumor colonies in a novel stirred mini-bioreactor.

The feasibility of a novel stirred bioreactor, the rotating aerial disk (RAD) design, was tested in this study. The novelty lies in its method of medium recirculation by convective airflow using a non-contact planar disc, a variation on a physically defined theoretical model. Computational predictions of improved oxygenation were confirmed by increases in measured dissolved oxygen, even at Reynolds numbers (100-200) where flow is mostly laminar. EL-4 mouse lymphoma cells grown for the first time as suspension cultures in the RAD bioreactor, were mechanically re-organization into dense, circular three-dimensional colonies (diameter 3-5 mm, thickness 5-800 microm), more rapidly than we have observed previously. Cell proliferation in the RAD vessels was similar to static cultures, although lactate production from glucose was significantly lower, suggesting a shift toward aerobic glycolysis. This possible reversal of the 'Warburg effect' was accompanied by a decrease in mitochondrial activity, perhaps reflecting a more quiescent cytoplasmic state. The RAD device may be useful as scalable, three-dimensional solid tumor model under more physiological conditions then static culture.

PEDF-derived synthetic peptides exhibit antitumor activity in an orthotopic model of human osteosarcoma.

Pigment epithelium-derived factor (PEDF) is one of the most potent inhibitors of angiogenesis, and has recently been demonstrated to have an important multifunctional role in tumor growth, invasion, and metastasis. However, relatively little is known of mechanisms through which PEDF exerts its antitumor activity. Therefore, with the aim of identifying potential functional epitopes specifically against osteosarcoma, we evaluated the bioactivity of four 25-mer synthetic PEDF-derived peptides (termed StVOrth-1, -2 -3, and -4) against a human osteosarcoma cell line, SaOS-2. We found that StVOrth-2 (residues 78-102) predominantly inhibited tumor cell proliferation, while StVOrth-3 (residues 90-114) markedly increased cellular adhesion to collagen type-1, with StVOrth-4 (residues 387-411) demonstrating most significant inhibition of Matrigel invasion. Furthermore, we show that StVOrth-1 (residues 40-64), -2 and -3 induce osteoblastic differentiation, evidenced by increased mineralized nodule formation. Interestingly, although no peptide inhibited angiogenesis in the tube formation assay, StVOrth-3 and -4 markedly suppressed VEGF expression. We further tested the activity of StVOrth-2 and StVOrth-3 in vivo, in an orthotopic model of osteosarcoma and found that both peptides significantly inhibited primary tumor growth and the development of pulmonary metastases. Together these results provide greater insight into the potential mechanisms through which PEDF exerts its antitumor function. Furthermore, this raises the possibility of developing short PEDF fragments as lead compounds for the treatment of osteosarcoma.

Chitosan microparticles encapsulating PEDF plasmid demonstrate efficacy in an orthotopic metastatic model of osteosarcoma.

The major stumbling block for most therapies against deep-seated disease, including tumours, is inefficient drug delivery. Such a concern is particularly important for osteosarcoma, the predominant form of bone cancer, and the largest cancer of its type in the paediatric age group. Pigment epithelium-derived factor (PEDF) is the most potent anti-angiogenic factor found endogenously in the body, with an increasing number of reports pointing to its direct antitumour activity. In this report, when a plasmid expressing PEDF (pPEDF) was encapsulated within two types of chitosan microparticles, anti-invasion and increased adhesion of the osteosarcoma cell line SaOS-2 was noted. Microparticles were formulated using two methods of complex coacervation and were approximately 400-600 nm in diameter. The plasmids were strongly attached to the particles which were polymorphic in shape as determined by electron microscopy. Preliminary experiments with the green fluorescent protein (GFP) reporter plasmid revealed that cells were efficiently transfected with the particles, with particles outlasting transfection with lipofectamine cationic liposomes at 5 days. In vivo, the better pPEDF microparticle resulted in a decrease in primary tumour growth, reduced bone lysis and reduced establishment of lung metastases in a clinically relevant orthotopic model of osteosarcoma. Thus, this new mode of localised gene delivery may hold promise for molecular therapy of osteosarcoma.

Inhibition of orthotopic osteosarcoma growth and metastasis by multitargeted antitumor activities of pigment epithelium-derived factor.

Osteosarcoma is major cause of cancer-related death in the pediatric age group, and this is due to the development of pulmonary metastases that fail to be eradicated with current treatment regimes. Although there have been significant improvements in the long-term survival of such patients, 25-50% with initially non-metastatic disease, subsequently develop metastases and this remains the major cause of death for these patients. In this study, we report the multimodal activity of pigment epithelium-derived factor (PEDF) in inhibiting osteosarcoma growth, angiogenesis and metastasis. In vitro, we found that administration of recombinant PEDF (rPEDF) on two osteosarcoma cell lines (rat UMR 106-01 and human SaOS-2) significantly reduced tumor cell proliferation and increased apoptosis, as well as decreased cell invasion, angiogenesis, and increased adhesion to collagen type-1. Administration of rPEDF upregulated the mRNA expression of phenotypic osteoblast differentiation markers (ALP, pro-alpha(1) collagen and osteocalcin) in a pre-osteoblastic cell line, UMR 201, and also increased mineralized nodule formation in both UMR 106-01 and SaOS-2. In vivo, rPEDF dramatically suppressed primary osteosarcoma growth and the development of macroscopic pulmonary metastases in an orthotopic model of human osteosarcoma (SaOS-2). Interestingly, no activity was seen in tumors grown subcutaneously, suggesting a paracrine interaction between PEDF and the bone microenvironment. Preliminary pharmacoevaluation studies demonstrated rPEDF stability within media containing serum and osteosarcoma cells, and no gross systemic toxicity was observed in vivo with rPEDF administration. These results suggest that PEDF is emerging as an attractive and clinically appealing drug candidate for the treatment of osteosarcoma.

A novel orthotopic murine model provides insights into cellular and molecular characteristics contributing to human osteosarcoma.

As a reliable model for osteosarcoma is lacking, three human cell lines (SaOS-2, U2OS and 143B) were evaluated in cell-based assays for proliferation, adhesion, migration, invasion, anchorage-independent growth, angiogenesis, mineralised nodule formation, plasmid transfection and oligonucleotide transfection. Tumor take and metastasis after orthotopic injection of the three cell lines into mice was monitored. The levels of expression of typical bone markers were determined with semi-quantitative RT-PCR in cultured cells, primary tumors, and for the SaOS-2 cell line, the metastases. Tumors grew and spread to the lungs within 3 and 5 weeks respectively, mimicking the clinical progression of the disease as analysed by x-ray. Expression of molecular markers in SaOS-2 indicated a mostly differentiated cell type at the primary and secondary sites. The ability of osteosarcoma cells to interact with collagen-1 and to form mineralised deposits correlated positively with tumor aggression in vivo. Expression of alkaline phosphatase was a common theme in both tumor models at the primary site. The newly established SaOS-2 model should allow the testing of candidate anti-osteosarcoma agents as well as dissection of more intricate mechanisms involved in human osteosarcoma.