Biomedical Engineering Reference
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minimal regeneration. The amount of 25 kDa PEI contained in each implant
scaffold is unknown, and there is no discussion on the possible toxicity of the PEI
delivered in the implant. A previous study from our lab found that small amounts
of PEI (16-32 mg) interfered with bone formation at an ectopic site following delivery
of BMP-2 albumin nanoparticles (Zhang et al.
2009
). Given that 0.2 mg of plasmid
DNA was delivered, more than 32 mg of PEI is expected to be required to condense
the plasmid DNA. Thus, some cytotoxicity is expected in these implants. Although
a ~4.5-fold increase in bone formation was detected over no plasmid and naked
plasmid controls, the majority of the defect still was not healed after 15 weeks with
PEI-condensed plasmid. When naked plasmid DNA or the scaffold alone was deliv-
ered, bone formation was only seen along the periphery, with no differences
between the two groups, suggesting differences in either cell penetration into the
scaffold or the regenerative capacity at each of the defect sites. The delivery of
0.5 mg of BMP-4 in collagen led to bridging of a femur defect by 9 weeks (Fang
et al.
1996
), while here 0.2 mg of BMP-4 delivered in a PLGA scaffold was not able
to show significant bone formation over a blank PLGA scaffold in a skull defect.
The HA, the main non-organic component of bone, and other calcium phos-
phates are commonly used scaffolds in bone regeneration, so that it is natural to
employ them for plasmid DNA delivery as well. An HA fiber scaffold loaded with
BMP-2 plasmid condensed in a CaP solution was implanted subcutaneously in rats
(Oda et al.
2009
). At 4 weeks, radiopague volume of the implants containing 50 or
100 mg of BMP-2 plasmid was higher than the HA scaffold alone. However, the HA
fibers themselves contained radiopaque regions detected by micro-CT as well, sug-
gesting that the scaffold itself may induce calcification even in the absence of thera-
peutic gene expression. By week 12, implants containing 10, 50 or 100 mg of
BMP-2 plasmid all had higher bone volume than the empty scaffold. The mineral
content of scaffolds containing 50 mg of BMP-2 plasmid maintained high levels
from week 4 to 12, and although there was no significant increase, there was no
decrease in mineral content, as was observed in other groups.
A similar study employed porous HA to deliver 10 mg of BMP-2 plasmid via
cationic liposomes, using the commercially available SuperFect™ transfection
reagent. The scaffolds were used to fill a rabbit critical-sized cranial defect (Ono
et al.
2004
). After 3 weeks, groups with the BMP-2 gene in liposomes showed new
bone formation on the bottom side of the scaffold. At 9 weeks, new bone tissue had
penetrated the lower half of the HA scaffold. Bone formation was also observed in
the absence of the HA scaffold when the BMP-2 plasmid liposomes were adminis-
tered directly to the site, and closed the defect by week 6. In total, 40 mg of the
liposome was administered to the site, an amount the authors have chosen based on
the desire to avoid cytotoxicity.
Finally, one group used employed as little as 1.3 mg of plasmid DNA (the lowest
amount known to the authors) and showed successful bone regeneration in a mouse
skull defect (Itaka et al.
2007
). A block polymer consisting of polyethyleneglycol,
aspartate and diethylenetriamine was used to deliver the plasmid DNA. Two genes
were delivered to the defect site: runt-related transcription factor 2 (Runx2) and
constitutively active form of activin receptor-like kinase 6 (caALK6), both
intracellular mediators involved in osteogenic differentiation. This is a unique
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