Collagen gel delivery of Tgf‐β₃ non‐viral plasmid DNA in rat osteoblast and calvarial culture

Abstract

Different forms of collagen as a carrier for naked plasmid DNA have shown potential as vehicles for therapeutic gene delivery and tissue engineering. The objective of this study was to determine the suitability of a dense collagen gel as a vehicle for sustained delivery of plasmid DNA in cell and organ culture. Plasmid DNA encoding Tgf-beta(3) was combined with collagen gel. DNA released into the media was measured by Pico-Green spectrophotometry. Results showed that DNA was released from the collagen gel at a gradual rate for up to 14 days. To evaluate collagen-mediated transfection in tissue, calvariae were exposed to collagen containing plasmid encoding GFP or DsRed. Transfection was visualized by fluorescence localized to tissue adjacent to the vehicle. To evaluate protein production, fetal rat calvarial osteoblasts were cultured with a collagen/Tgf-beta(3) plasmid mixture or in media containing plasmid alone. Media was collected at various time points to measure Tgf-beta(3) protein production. ELISA assays showed that collagen-transfected osteoblasts demonstrated an elevated Tgf-beta(3) protein production for up to 14 days. Therefore, collagen delivery of viable plasmid DNA created a sustained transient transfection of calvarial osteoblasts resulting in prolonged and elevated growth factor production. Together, these results suggest that use of collagen gel as a vehicle may provide a strategy to achieve localized and controlled, non-viral gene delivery in vivo.

Comparison of Craniofacial Phenotype in Craniosynostotic Rabbits Treated with Anti–Tgf-β2 at Suturectomy Site

“…Humans achieve approximately 90% of their total brain growth by 2 years of age (Enlow, 1990), compared with rabbits requiring just a few weeks. In humans, therapeutic agents would have to be delivered in multiple doses using existing technologies (Boyan et al, 1999;Alsberg et al, 2001Alsberg et al, , 2002Franceschi, 2005;Premaraj et al, 2005) or via a novel, slow-release vehicle that would last long enough to have a therapeutic effect on neurocranial growth.…”

Controlled Release Scaffolds for Bone Tissue Engineering

“…It is also thought to play a role in regulating cell adhesion and extracellular matrix formation. Premaraj et al27 have also made use of a collagen gel to deliver TGF‐beta stimuli. In this study, 5 mg of TGF‐beta3 plasmid DNA was mixed 1:1 with a collagen gel (32 mg/mL, NeuColl, Angiotech Pharmaceuticals Inc., Vancouver, Canada).…”

Tissue-engineered trachea: History, problems and the future☆

“…Meanwhile, many promising experiments suggest the possibility of GFs gene delivery. Unfortunately in these experiments, both viral method (transduction) and nonviral method (transfection) showed technical limitations: the former often goes with likelihood of mutagenesis, carcinogenesis and immune response to viral infection or viral proteins while the latter still suffers from low efficiency of gene delivery into the target cell population [64][65][66][67][68]. The tragic death of a patient due to massive cytokine release gives rise to heated ethical debate in trial gene therapy recently.…”