Gene Therapy for the Enhancement of Fracture Healing - Engineered Bone: Tissue Engineering Intelligence Unit - page 139

Figure 4. Delivery of BMP-4 and PTH 1-34 cDNA via plasmid vectors embedded in a collagen sponge
results in bone formation in a rat femur critical-sized defect model. a) Plain x-ray film of control rat femur
osteotomy 9 weeks post-surgery showing no healing of the defect. b) Plain x-ray film showing new bone
bridging the gap of a 5-mm defect four weeks post-implantation. c) The same gap as in (b) without the
external fixator, 17 weeks post-surgery. Arrows point to sites of original defect which is now filled with radio-
dense tissue in (b) and (c). Plasmid delivery of BMP-4 and PTH 1-34 cDNA in a collagen sponge was able
to heal a rat femur critical-sized defect.
the wound site. Examples of initial animal studies examined the use of both plasmid and viral
vectors to deliver osteoinductive growth factors to defect sites in bone.
BMP-4 and PTH 1-34 cDNA was delivered to the site of 5 mm midshaft osteotomies in
rats using direct gene transfer with a plasmid vector. 128 A collagen sponge served as the carrier
matrix for the plasmid DNA. New bone formation was stimulated at the site of the defect
where in controls, fibrous tissue formed. Bone induction resulted in functional union of large
segmental defects in the rat femur (Fig. 4). The rates of new bone formation paralleled results
from previous studies which used direct protein therapy to administer BMP-2, OP-1 and TGF- β
to the wound site. 165,166 Therefore, plasmid DNA can be used to transfer growth factor cDNA
to repair cells at the wound site. The amount of new bone formation is comparable to that
achieved using direct delivery of large doses of protein. This represents a simple way to simulate
mammalian bone growth by controlling transient, local, over-expression of osteogenic factors.
Further, gene transfer may be an advantageous strategy for drug delivery for molecules that
normally require post-translational modifications. A relevant example is TGF- β , which has
been difficult to develop as a recombinant bone growth therapy despite many in vivo studies
supporting its role in osteogenesis. Given the large number of known osteoinductive factors,
others should be examined for gene therapy strategies either alone or in combination. The
method of delivery for each factor depends on its function, expression level requirements, and
mechanism of action. Further, since plasmid vectors are known to have relatively low gene
transfer efficiency, the use of viral vectors for the delivery of osteoinductive growth factors is
often warranted.
Adenoviral vectors have also been used to deliver growth factors to a site of fractured bone.
By directly injecting adenoviral vectors into rabbits, in vivo gene therapy provides a minimally
invasive treatment with little damage to local blood circulation. Marker gene transfer (LacZ)
was shown to persist locally for up to six weeks at a declining rate. 41 For the treatment of
complications following fracture, this duration may be suitable. It was also shown that bone
marrow cells, expressing BMP-2 via viral vector can heal a segmental femoral defect. 167 The
results show the feasibility of an ex vivo gene therapy technique with short-term cultures of
bone marrow cells. These studies lead to perhaps the most exciting results; those in which gene
therapy enhance tissue engineering approaches to bone healing.
Cultured rabbit periosteal cells transduced retrovirally with BMP-7 cDNA were shown to
induce new bone formation and repair bone defects. Further, TGF- β cDNA can be effectively
transferred to osteoblasts and osteocytes in vivo. This treatment can be administered percuta-
neously. These techniques may be superior to the use of growth factor proteins alone; overcom-
ing problems of protein instability, localization and duration of expression. Local gene therapy