Gene Therapy Induced Surgical Revascularization of Cryopreserved Allogenic Bone: In a Yucatan Minipig Model

INTRODUCTION:

Current surgical treatment options for segmental bone often include the use of cryopreserved allogeneic banked bone closely matched in size and shape to the resected specimen. Structural allografts provide immediate stability, but remain largely necrotic with time, resulting in significant risks of infection, non-union and late stress fracture. Restoring the vitality of allograft bone  may resolve many of these complications. We have tested a means to accomplish this goal in a Yucatan minipig segmental tibial defect model. At the time of defect reconstruction with a matched cryopreserved allograft, a cranial tibial arteriovenous (AV) bundle was implanted, with and without endothelial transfection with vasculogenic growth factors within the medullary canal.After a survival period, we assessed healing, measured bone viability and quantified bone remodeling.

 

METHODS:

Segmental defects of 3.5 cm were created in 16 Yucatan minipig tibias, and restored using cryopreserved allogeneic bone and double plating. In all 16 pigs the anterior tibial artery and vein were ligated distally and cut. The AV-bundle was placed within the medullary canal. In 8 of the pigs the AV-bundle was transfected with VEGF and PDGF (VEGF-group)  using adeno associated virus (AAV) as the transfecting vector. The other8 pigs had no growth factors added to their AV-bundle (control group). After 20 weeks the pigs were sacrificed and the transplanted allografts were analyzed. The contralateral sides were used as normal controls. Vascular volume was calculated as a measurement for revascularization using micro-CT after both femoral arteries were injected with microfil. The bone formed between two fluorochrome lables ( tetracycline  and calceine) administered 14 and 4 days prior to sacrifice was studied using Sanderson’s rapid bone stains.We measured osteoblasts as well as osteoid-covered surface (bone formation) as well as  osteoclasts, and eroded surfaces (bone resorption/remodeling).

 

RESULTS:

The vascular volume in the VEGF and PDGF-treated group (164 mm3) was significantly higher compared to the control group (88 mm3, p=0,003) and the untreated contralateral sides (36 mm3, p=0,016). The inner cortex showed significantly more bone remodeling in the VEGF and PDGF treated group compared to the control group (Bone Formation Rate: 557 μm3/ μm3/jaar versus 403 μm3/μm3/jaar, p=0,013). Compared to the untreated contralateral side the VEGF-group showed no significant difference (Bone Formation Rate: 557 μm3/μm3/jaar versus 80 μm3/μm3/ jaar, p=0,109). The Sanderson’s rapid bone stains showed significant higher numbers for osteoblasts in the inner cortex (224) compared to the control group (119, p=0.007), osteoid surface (45mm2 versus 26mm2, p=0.015) and eroded surface (11mm2 versus 5mm2, p=0.015), but not for osteoclast number (7 versus 7, p=0.800).

CONCLUSION:

Revascularization of cryopreserved segmental tibia through placement of an AV-bundle intramedullary and adding growth factors VEGF and PDGF results in increased neoangiogenesis and bone formation compared to the use of the AV-bundle alone in a Yucatan minipig model.