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Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_II | Pages 341 - 342
1 May 2006
Hasharoni A Azoulay T Zilberman Y Liebergall M Gazit D
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Introduction: Spinal fusion has become a popular surgical technique. Problems of fusion failure or pseudo-arthrosis as well as bone graft donor site complications are common. Ex vivo gene therapy using mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP) genes can provide a local supply of precursor cells and a supra-physiological dose of osteoinductive molecules that may promote bone formation and lead to spinal fusion.

Methods: Thirty 6–7 weeks old C3H/HeN immune-competent female mice received an injection of 2x106 genetically engineered MSCs to the para-vertebral muscle of the lumbar spine (L2-L6) under manual palpation. Ten animals served as negative control group and 20 animals constituted the experimental group.

Bone formation in the para spinal region of the injected animals was evaluated by histology staining. Quantitative analysis of the fusion mass was monitored by micro computerized tomography (μCT).

Results: At 1, 2, 4 and 8 weeks post injection. Bone formation was extensive, as soon as the 1st week post injection, in the area adjacent to and adhering to the posterior elements of the spine in all the study animals. None of the control animals, in which hBMP-2 was inhibited, showed any new bone formation.

Discussion: Exogenously regulated expression of the hBMP-2 enabled us to regulate bone formation in vivo, using genetically engineered MSC system. The effect of hBMP-2 in inducing bone formation was monitored in real time, non-invasive and quantitative system that enabled us to better understand the biological process during bone regeneration and repair. Our data demonstrate a regulated and monitored system for inducing bone for spinal fusion. We conclude that controlled gene therapy for spinal fusion can be achieved using Tet-regulated hBMP-2 gene and MCSs.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_III | Pages 391 - 391
1 Sep 2005
Loebenberg M Pelled G Hoffman A Zilberman Y Shinar H Keinan-Adamsky K Navon G Gross G Gazit D
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Introduction: New biotechnologies create opportunities for gene therapy to promote rotator cuff healing. We have previously demonstrated that genetically engineered mesenchymal stem cells (MSCs) over expressing BMP-2 and SMAD8 signaling molecule differentiate to tenocytes in vitro and in vivo. Therefore, we hypothesized that rotator cuff defect could be regenerated using genetically engineered MSCs.

Method: Nonviral methods were utilized to establish genetically engineered MSCs that co-express BMP-2 and the Smad8 signaling molecule. A previously validated animal model was utilized to examine rotator cuff healing. A 2mm x2mm full thickness defect was created in the infraspinatus tendon of 8 nude rats. A collagen-I biomembrane (TissueMend) containing 3 x 106 engineered cells was sewn into the defect. An identical control procedure was repeated on the contralateral side with biomembrane containing non-engineered MSCs.

Results: 4 weeks post implantation the area of implantation was isolated and analyzed by light microscopy and histochemical staining. Analysis of the engineered implants revealed the formation of dense connective tissue with parallel-organized fibers and spindle shaped cells, unlike the control samples. Proton Double Quantum Filtered Magnetic Resonance Imaging technique of the rotator cuff tendons demonstrated an increased presence of organized collagen fibers within the engineered rotator cuff tissue when compared with either native rotator cuff or those treated with non-engineered MSCs.

Conclusion: This is the first report showing rotator cuff tendon repair using genetically engineered MSCs. Moreover these findings may have considerable importance for tendon healing and may indicate a clinical gene therapy platform to augment surgical repair.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_III | Pages 316 - 316
1 Nov 2002
Turgeman G Liebergall M Zilberman Y Pelled G Aslan H Peyser A Gazit Z Domb A Gazit D
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Mesenchymal Stem Cells (MSCs) are key regulators in senile osteoporosis and in bone formation and regeneration. MSCs are therefore suitable candidates for stem cells mediated gene therapy of bone. Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) is a highly osteoinductive cytokine, promoting osteogenic differentiation of MSCs.

We hypothesized that genetically engineered MSCs, expressing rhBMP2, can be utilized for targeted cell mediated gene therapy for local and systemic bone disorders and for bone/cartilage tissue engineering. Engineered MSCs expressing rhBMP-2 have both autocrine and paracrine effects enabling the engineered cells to actively participate in bone formation.

We conditionally expressed rhBMP2 (tet-controlled gene expression, tet-off system) in mouse and human mesenchymal stem cells. RhBMP2 expressing clones (tet-off and adeno-BMP2 infected MSCs), spontaneously differentiated into osteogenic cells in vitro and in vivo.

Engineered MSCs were transplanted locally and tracked in vivo in radial segmental defects (regenerating site) and in ectopic muscular and subcutaneous sites (non-regenerating sites). In vitro and in vivo analysis revealed rhBMP2 expression and function, confirmed by RT-PCR, ELISA, western blot, immunohistochemistry and bioassays. Secretion of rhBMP2 in vitro was controlled by tetracycline and resulted in secretion of 1231 ng/24 hours/106 cells.

Quantitative Micro-CT 3-Dimentional reconstruction revealed complete bone regeneration regulated by tetracycline in vivo, indicating the potential of this platform for bone and cartilage tissue engineering. Angiogenesis, a crucial element in tissue engineering, was increased by 10-folds in transplants of rhBMP2 expressing MSCs (tet-off), shown by histomorphometry and MRI analysis (p< 0.05). In order to establish a gene therapy platform for systemic bone disorders, MSCs with tet-controlled rhBMP-2 expression, were injected systemically (iv).

These engineered MSCs were genetically modified in order to achieve homing to the bone marrow. Systemic non invasive tracking of engineered MSCs was achieved by recording topographical bioluminescence derived from luciferase expression detected by a coupled charged CCD imaging camera. For clinical situations that require immuno-isolation of transplanted cells, we developed an additional platform utilizing cell encapsulation technique. Immuno-isolated engineered MSCs, with tet-controlled rhBMP-2 expression, encapsulated with sodium alginate induced bone formation by paracrine effect of secreted rhBMP-2. Finally, we have characterized a novel tissue-engineering platform composed of engineered MSCs and biodegradable polymeric scaffolds, creating a 3D bone tissue in rotating Bioreactors. Our results indicate that engineered MSCs and polymeric scaffolds can be utilized for ex vivo bone tissue engineering. We therefore conclude that genetically engineered MSCs expressing rhBMP-2 under tetracycline control are applicable for: a) local and systemic gene therapy to bone, and b) bone tissue engineering. Our studies should lead to the creation of gene therapy platforms for systemic and local bone diseases in humans and bone/cartilage tissue engineering.