Gene Therapy in Bone Regeneration: A Summary of Delivery Approaches for Effective Therapies - Intracellular Delivery: Fundamentals and Applications - page 833

beneficial effects were articulated before. Whereas the use of DNA-condensing
carriers seems to be paramount for effective gene expression, in vivo attempts for
bone regeneration have occasionally relied on plasmid DNA delivery with scaffold
alone without the appropriate carriers. Figure 4 summarizes the reported attempts
at non-viral gene delivery for bone regeneration.

4.1 Plasmid DNA Delivery Without Carriers

One can avoid the use of carriers and rely on electroporation for cellular uptake of
genes; the feasibility of this approach was shown in a rat subcutaneous model
where radiopague bone was obtained after the delivery of 25-50 mg BMP-2/-7
plasmid to a rat ectopic site (Kawai et al. 2009 ). However, this physical method to
disrupt plasma membranes may leave too much tissue trauma, as dystrophic calci-
fication has been seen in controls (Kishimoto et al. 2002 ) when 100 mg of BMP-4
plasmid was delivered ectopically to rat muscle using electroporation. Additionally,
it may be difficult to control to where the plasmid DNA is delivered, especially in
intraosseous applications where the bone is well-surrounded of muscle and soft
tissue. Plasmid DNA encoding for OP-1 was also delivered by a collagen scaffold
without a specific carrier (Bright et al. 2006 ). Histological bone formation was
evident with the use of the collagen carrier but no radio-opaque bone formation was
observed even with 500 mg of plasmid, indicating less than efficacious bone induc-
tion from a clinical perspective. No bone formation was detected histologically in
that study when naked plasmid DNA was delivered, supporting the role of collagen
in localizing or retaining the plasmid DNA. It was not known if collagen condensed
the plasmid DNA into a nanoparticle for cell uptake, or simply retained the plasmid
DNA in its native configuration at the site for a prolonged time.
More success was seen with a collagen scaffold delivering a plasmid coding for
human parathyroid hormone 1-34 (PTH1-34) in a canine tibia critical defect model
(Bonadio et al. 1999 ). The plasmid was added to a collagen solution, which was
frozen and lyophilized as a unitary device. A 25% increase in bone mass was seen
with 40 mg of plasmid DNA after 4 weeks, and the majority of the bone gap was
filled at 6 weeks with 100 mg of plasmid. While this model shows good promise
for clinical utility, the high amount of plasmid (100 mg) required for healing this
tibial defect leaves room for improvement. Another study investigated a combina-
tion of therapeutic genes delivered by a collagen sponge to a rat critical defect
femur model (Fang et al. 1996 ). Only fibrous tissue was seen in control groups;
however the delivery of BMP-4 plasmid resulting in new bone forming to bridge
the defect at 9 weeks and sufficient new bone had formed by 18 weeks to remove
external fixation supporting the defect. Co-delivery of a BMP-4 and PTH1-34
expression plasmid was found to be superior to BMP-4 alone. The combination of
BMP-4 and PTH1-34 led to accelerated healing, where defect was bridged at
4 weeks, and external fixation could be removed at 12 weeks, but even this favour-
able configuration still required 0.5-1 mg of plasmid DNA. Since a group looking