title: Synthetic hybrid grafts for craniofacial reconstruction: sustained gene delivery using a calcium phosphate bone mineral substitute.
abstract: These experiments were performed to evaluate the efficacy of a biocompatible bone cement, Norian CRS, engineered as a hybrid graft for simultaneous bone matrix reconstruction and sustained, site-directed gene transfer using an adenoviral vector. Norian CRS was cured ex vivo by mixing a calcium source powder with a phosphate source solution to form a paste. To 1.0 ml of the cement was added 50 microl of a solution containing 1 x 10(8) plaque-forming units of a replication-deficient adenoviral vector containing a bacterial beta-galactosidase reporter gene (AdLacZ). In vitro, fragments of the hybrid Norian-AdLacZ construct were placed into 12-microm-pore culture plate inserts and cocultured with human fibroblasts. The same insert was transferred to a new well of fibroblasts every 48 hours for 30 days, and, after allowing 72 hours for gene expression, fibroblasts were examined for transgene expression by 5 bromo-4-chloro-3-indoyl-beta-D-galactosidase (X-gal) staining. In vivo, the Norian-AdLacZ hybrid was implanted into 10-mm frontal bone defects in 3-week-old piglets. The implant sites were harvested after 5 days and were examined for transgene expression by X-gal staining. X-gal staining of fibroblasts incubated with the hybrid Norian-AdLacZ construct was observed throughout the 30-day period. Transgene expression was also observed about the periphery of the calvarial defects treated with hybrid Norian-AdLacZ constructs. Thus, adenoviral vectors may be incorporated successfully into a synthetic calcium phosphate bone mineral substitute to provide effective, sustained local gene delivery.
authors.affiliation: Department of Surgery, The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine, 19104-4318, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Kirschner
authors.forename: J
authors.initials: J
authors.lastname: Karmacharya
authors.forename: G
authors.initials: G
authors.lastname: Ong
authors.forename: O
authors.initials: O
authors.lastname: Hunenko
authors.forename: J E
authors.initials: JE
authors.lastname: Losee
authors.forename: B
authors.initials: B
authors.lastname: Martin
authors.forename: T M
authors.initials: TM
authors.lastname: Crombleholme
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001672:Biocompatible Materials; D018786:Bone Substitutes; D002130:Calcium Phosphates; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D020007:Implants, Experimental; D012886:Skull / Q000601:surgery*; D013552:Swine; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001672:Biocompatible Materials; D018786:Bone Substitutes; D002130:Calcium Phosphates; D017077:Culture Media, Conditioned; D001616:beta-Galactosidase