Original articleTendonTendon Healing In Vitro: bFGF Gene Transfer to Tenocytes by Adeno-Associated Viral Vectors Promotes Expression of Collagen Genes
Section snippets
Construction of Adeno-Associated Viral 2–Basic Fibroblast Growth Factor Gene Delivery Unit
Adeno-associated viral 2–basic fibroblast growth factor and AAV2-lacZ vectors were used in this study. The AAV2-bFGF vector contains the rat bFGF gene (X07285; Genebank, National Institutes of Health, Bethesda, MD) and has a nuclear localization signal under the regulation of the cytomegalovirus immediate early promoter.16 A segment containing bFGF gene was cut through EcoRI and XhoI restriction sites of a plasmid that preserved rat bFGF gene. This segment was cloned to another vector
Confirmation of Efficiency of Gene Delivery by Adeno-Associated Viral Vectors
X-gal staining of lacZ-transduced cells showed blue nuclei. Microscopic observation of blue-stained nuclei confirmed that AAV2 could deliver the exogenous gene at the concentration of viral particles used in this study (MOI = 100) into tenocytes (Fig. 2). The transduction rate of AAV2 lacZ was between 5% and 10% in tenocytes.
Expression of the Basic Fibroblast Growth Factor Genes
Levels of expression of the bFGF gene from the tenocytes treated with AAV2-bFGF were significantly higher than expression of the bFGF gene in the controls (p < .001) (
Discussion
Flexor tendons, particularly those in the intrasynovial area, lack sufficient cellularity and have low growth factor levels, which are the basic reasons that adhesions or ruptures may occur after surgery. Delivery of growth factors to proliferating tenocytes in vitro markedly enhanced the proliferation rate and collagen production.12, 13, 14, 15 Gene therapy may provide the tendons with genes to produce growth factors over a critical period of healing. The delivery of a growth factor gene
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