The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats.
METHODS
Rat spinal fusion model.


OBJECTIVE
This study aimed to assess the ability of rat bone marrow cells (RBMCs) transfected with bone morphogenetic protein (BMP)-2-containing lentivirus to induce a posterolateral spinal fusion in a rat model.


BACKGROUND
Spinal arthrodesis is a commonly performed spinal procedure and autograft remains the standard for achieving spinal fusion. However, its procurement is associated with significant morbidity, and the rate of pseudoarthrosis has been reported to be 5% to 43%. Nonunion frequently leads to an unsatisfactory resolution of clinical symptoms and usually results in high medical costs and morbidity as well as the need for additional surgeries. These problems have led surgeons to search for alternative solutions to stimulate bone formation. Recombinant BMPs have also been used successfully in clinical trials. However, large doses of BMPs were required to induce adequate bone repair. The development of a regional gene therapy may be a more efficient method to deliver proteins to a specific anatomic site. Furthermore, adeno-BMP-2-producing rat bone marrow-derived cells have been used successfully to induce posterior spinal fusion. Recently, lentiviral vectors on the basis of human immunodeficiency virus have been developed for gene therapy. Lentiviruses are capable of insertion into the host genome, ensuring a prolonged gene expression. However, safety issues are a major concern when adopting these vectors for clinical use.


METHODS
In vitro study, we used RBMCs transfected with lentivirus vectors encoding BMP-2 (Lenti-BMP-2), RBMCs transfected with lentivirus vectors encoding the green fluorescent protein (GFP) (Lenti-GFP), and untransfected RBMCs; the latter 2 were used as controls. Alkaline phosphatase (ALP) staining and ALP activity were compared between the groups to assess the ability of the Lenti-BMP-2-transfected RBMCs to stimulate osteoblastic differentiation. In the rat posterolateral spine fusion model, the experimental study comprised 4 groups. Group 1 comprised 6 animals that were implanted with a collagen sponge containing 5 million RBMCs transfected with Lenti-BMP-2. Group 2 comprised 3 animals that were implanted with a collagen sponge containing 5 million RBMCs transfected with Lenti-GFP. Group 3 comprised 6 animals that were implanted with a collagen sponge containing 5 million untransfected RBMCs. Group 4 comprised 3 animals that were implanted with a collagen sponge alone. The rats were assessed by radiographs obtained at 4, 6, and 8 weeks. After death, their spines were explanted and assessed by manual palpation, high-resolution microcomputerized tomography, and histologic analysis.


RESULTS
The ALP staining was significantly greater in the Lenti-BMP-2-transfected RBMCs than in the untransfected RBMCs and the Lenti-GFP-transfected RBMCs. The ALP activity was 3-fold greater in the Lenti-BMP-2-transfected RBMCs than in the untransfected RBMCs and the Lenti-GFP-transfected RBMCs. In the rat spine fusion model, radiographic evaluation, high-resolution microcomputerized tomography, and manual palpation revealed spinal fusion in all the rats in Group 1 at 8 weeks. Groups 2, 3, and 4 comprised the control group. None of the rats in the control group (0 of 12) developed fusion at L4-L5.


CONCLUSIONS
The present study demonstrated that BMP-2-producing RBMCs, created through lentiviral gene transfer, induced sufficient spinal fusion. The use of lentiviral vectors that contain the cDNA for BMP-2 will be a novel and promising approach for a spinal fusion strategy.
Miyazaki
Masashi
M
Department of Orthopaedic Surgery, University of California at Los Angeles, Los Angeles, CA 90095, USA. miyazakisora@gmail.com
Sugiyama
Osamu
O
Tow
Benjamin
B
Zou
Jun
J
Morishita
Yuichiro
Y
Wei
Feng
F
Napoli
Antonia
A
Sintuu
Chananit
C
Lieberman
Jay R
JR
Wang
Jeffrey C
JC
D000818:Animals; D016026:Bone Marrow Transplantation / Q000379:methods*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis / Q000235:genetics*; D001861:Bone Regeneration / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods*; D005822:Genetic Vectors / Q000009:adverse effects / Q000235:genetics / Q000627:therapeutic use; D007091:Image Processing, Computer-Assisted; D016086:Lentivirus / Q000235:genetics*; D008159:Lumbar Vertebrae / Q000601:surgery; D008297:Male; D023421:Models, Animal; D010012:Osteogenesis / Q000235:genetics; D011183:Postoperative Complications / Q000517:prevention & control; D051381:Rats; D011917:Rats, Inbred Lew; D013123:Spinal Fusion / Q000379:methods*; D013131:Spine / Q000601:surgery; D014057:Tomography, X-Ray Computed; D014162:Transfection / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics*; D016896:Treatment Outcome
C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta