B Sosnowski | PubMed Health

PubMed2005

Bone induction by AdBMP-2/collagen implants.

BACKGROUND: Demineralized bone matrix and recombinant human bone morphogenetic protein-2 or 7 (BMP-2 or BMP-7)-containing collagenous matrix have been shown to induce new bone formation in orthotopic and heterotopic sites. We examined the ability of subcutaneous implants of collagen combined with adenoviral vector containing the BMP-2 gene (AdBMP-2) to induce bone formation in rats. We also evaluated whether targeting the AdBMP-2 vector through an alternative receptor pathway, fibroblast growth factor (FGF), would increase the vector's potency. METHODS: In a time-course study, rat subcutaneous sites were implanted with (1) AdBMP-2 in rat-bone-derived collagen or (2) rat-bone-derived collagen alone. Samples were collected three, seven, fourteen, or thirty-five days after treatment. In a dose-response study, bone induction by AdBMP-2 in collagen (AdBMP-2/collagen) or by AdBMP-2 and FGF2 Fab' anti-adenovirus knob protein antibody in collagen (FGF2-AdBMP-2/collagen) was tested at fourteen days. Viral vector doses of 1 x 10(9) PN (viral particle number), 3 x 10(9) PN, 1 x10(10) PN, 3 x 10(10) PN, or 1 x 10(11) PN per implant were used. Equal amounts of collagen (25 mg) were used to formulate all implants. Explanted tissues were evaluated histologically to determine bone formation, specific activity of alkaline phosphatase, and calcium content. RESULTS: AdBMP-2/collagen implants induced robust bone formation. New bone was formed by the fourteenth day after implantation. In contrast, little or no bone was induced by the implant containing collagen alone. FGF2-AdBMP-2/collagen implants stimulated significantly more bone formation (p < 0.05) than did AdBMP-2/collagen implants, regardless of the dose of viral particles. CONCLUSIONS: Local delivery of AdBMP-2 in a collagen matrix rapidly induces bone formation, and targeting the virus through FGF receptors enhances the osteogenic potential of AdBMP-2.

Adenoviridae↗

PubMed1996

Large-scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin.

In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rFGF-2-SAP was cloned into the inducible pET 11a expression vector and transformed into Escherichia coli strain BL21 (DE3). The transformants were grown using a fed-batch fermentation until the A600 reached 85. At this stage, induction of the expression of the fusion protein led to the production of approximately 2.2 mg/liter per A600 unit. The soluble mitotoxin was purified to homogeneity from cell lysates via expanded bed adsorption chromatography followed by cation-exchange, heparin-affinity, and size-exclusion chromatography. Purified rFGF-2-SAP contained less than 0.5 EU/mg of endotoxin, as determined by gel clot analyses. The highly purified rFGF-2-SAP retained the toxin's ability to inhibit protein synthesis as measured in a cell-free system and was cytotoxic to a number of normal and neoplastic cell lines bearing FGF receptors. Binding studies establish that the fusion protein exerts its effects via the FGF high-affinity receptor.

Binding, Competitive↗