title: In vivo induction and delivery of nerve growth factor, using HEK-293 cells.
abstract: Tissue-engineering strategies offer hope to patients facing functional impairment after nerve injury. We have previously demonstrated that HEK-293 cells can release nerve growth factor (NGF) in vitro, using an inducible system of expression. In this study, our objective was to assess the efficacy of the NGF delivery system in vivo, using nude rats. HEK-293 cells were transfected with human NGF cDNA. Ponasterone A (PonA) was used as the inducing agent. NGF collection chambers were implanted subcutaneously in nude rats. Sealed chambers were filled with one of the following: (1) DMEM, (2) untransfected 293 cells (EcR-293) plus PonA, (3) untransfected EcR-293 without PonA, (4) transfected 293 cells (hNGF-EcR-293) plus PonA, or (5) transfected hNGF-EcR-293 without PonA. Chambers were aspirated 24, 48, and 120 h postimplantation. NGF secretion was analyzed in the following ways: (1) NGF protein expression bioactivity was assessed in a PC-12 cell bioassay, and (2) the concentration of secreted NGF was quantified by NGF ELISA. NGF quantification by ELISA reached a maximal release of 12.9 +/- 3.57 ng/mL at 120 h. PC-12 cells exposed to media from induced transfected HEK-293 cell chambers demonstrated higher levels of differentiation compared with controls. We conclude that hNGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent PonA. This regulated delivery system can secrete bioactive NGF for up to 5 days in vivo. We believe this regulated delivery system will be useful for tissue-engineered nerve constructs.
authors.affiliation: Aesthetic and Plastic Surgery Institute, University of California, Irvine, Orange, California 92868-3298, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: McConnell
authors.forename: Sanjay
authors.initials: S
authors.lastname: Dhar
authors.forename: Sanjay
authors.initials: S
authors.lastname: Naran
authors.forename: Thang
authors.initials: T
authors.lastname: Nguyen
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Bradshaw
authors.forename: Gregory R D
authors.initials: GR
authors.lastname: Evans
mesh_terms: D000818:Animals; D002460:Cell Line; D016503:Drug Delivery Systems / Q000379:methods*; D004343:Drug Implants; D005260:Female; D006801:Humans; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D016030:Kidney Transplantation / Q000379:methods*; D020932:Nerve Growth Factor / Q000008:administration & dosage* / Q000378:metabolism*; D009416:Nerve Regeneration / Q000187:drug effects; D051381:Rats; D011923:Rats, Nude; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism; D012584:Sciatic Nerve / Q000187:drug effects* / Q000293:injuries* / Q000473:pathology / Q000601:surgery; D016896:Treatment Outcome
chemical_list: D004343:Drug Implants; D011994:Recombinant Proteins; D020932:Nerve Growth Factor