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LMP-1 Retroviral Gene Therapy Influences Osteoblast Differentiation and Fracture Repair: A Preliminary Study

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Abstract

LIM mineralization protein-1 (LMP-1) is a novel intracellular osteogenic factor associated with bone development that has been implicated in the bone morphogenetic protein (BMP) pathway. This preliminary study evaluated the possibility of LMP-1-based retroviral gene therapy to stimulate osteoblast differentiation in vitro and fracture repair in vivo. A Moloney leukemia virus (MLV)-based retroviral vector to express LMP-1 with a hemagglutinin (HA) tag was developed, and its effects were evaluated on MC3T3-E1 cell differentiation and in the rat femur fracture model. MC3T3-E1 osteoblasts transduced with the MLV-HA-LMP-1 vector demonstrated significantly increased osteoblast marker gene expression (P < 0.05) and mineral deposition compared to control transduced cells. Femoral midshaft fractures were produced in Fischer 344 rats by the three-point bending technique. The MLV-HA-LMP-1 or control vector was applied at the fracture site through percutaneous injections 1 day postfracture. Analysis of fracture healing of 10 MLV-HA-LMP-1-treated and 10 control MLV-β-galactosidase (β-gal)–treated animals was completed at 3 weeks by X-ray, peripheral quantitative computed tomography, and histology. MLV-HA-LMP-1-treated animals had 63% more bone mineral content at the fracture site (P < 0.01), 34% greater total hard callus area (P < 0.05), and 45% less cartilage in the fracture callus (P < 0.05) compared to MLV-β-gal-treated animals. There was no effect of LMP-1 treatment on the density of the hard callus. Immunohistochemistry revealed expression of the LMP-1 transgene in the fracture callus at 21 days postfracture. Immunohistochemistry also revealed that LMP-1 transgene expression did not result in an increase in BMP-4 expression in the fracture callus. Compared to MLV-BMP-4 gene therapy studies, MLV-HA-LMP-1 gene therapy improved bony union of the fracture gap to a greater extent and did not cause heterotopic bone formation. This suggests that LMP-1 may be a better potential candidate for gene therapy for fracture repair than BMP-4. These exciting, albeit preliminary, findings indicate that LMP-1-based gene therapy may potentially be a simple and effective means to enhance fracture repair that warrants further investigation.

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Acknowledgements

The authors thank Dr. David J. Baylink for establishing the Gene Therapy Group at the Loma Linda VA Medical Center; Dr. Gordon Gill for providing the Enigma cDNA clone for the development of a retroviral vector for these studies; and Ryan Porte, Nancy Lowen, and Audrey Delgado for excellent technical support. This research was funded by a VA Merit Award, a Musculoskeletal Disease Center Congressional Appropriation, and the Medical Scientist Training Program at Loma Linda University.

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Authors and Affiliations

  1. Musculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, 11201 Benton Street, Loma Linda, CA, 92357, USA

    Cassandra A. Strohbach, Charles H. Rundle, Jon E. Wergedal, Shin-Tai Chen, Thomas A. Linkhart, K.-H. William Lau & Donna D. Strong

  2. Department of Medicine, Loma Linda University, Loma Linda, CA, 92354, USA

    Cassandra A. Strohbach, Charles H. Rundle, Jon E. Wergedal, Shin-Tai Chen, K.-H. William Lau & Donna D. Strong

  3. Department of Biochemistry and Microbiology, Loma Linda University, Loma Linda, CA, 92354, USA

    Cassandra A. Strohbach, Jon E. Wergedal, Shin-Tai Chen, Thomas A. Linkhart, K.-H. William Lau & Donna D. Strong

  4. Department of Pediatrics, Loma Linda University, Loma Linda, CA, 92354, USA

    Thomas A. Linkhart

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  1. Cassandra A. Strohbach
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  2. Charles H. Rundle
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  6. K.-H. William Lau
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  7. Donna D. Strong
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Correspondence to Donna D. Strong.

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Strohbach, C.A., Rundle, C.H., Wergedal, J.E. et al. LMP-1 Retroviral Gene Therapy Influences Osteoblast Differentiation and Fracture Repair: A Preliminary Study. Calcif Tissue Int 83, 202–211 (2008). https://doi.org/10.1007/s00223-008-9163-0

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  • DOI: https://doi.org/10.1007/s00223-008-9163-0

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