Elsevier

The Journal of Hand Surgery

Volume 30, Issue 6, November 2005, Pages 1255-1261
The Journal of Hand Surgery

Original article
Tendon
Tendon Healing In Vitro: bFGF Gene Transfer to Tenocytes by Adeno-Associated Viral Vectors Promotes Expression of Collagen Genes

https://doi.org/10.1016/j.jhsa.2005.06.001Get rights and content

Purpose

Adeno-associated virus-mediated gene transfer is promising in the delivery of genes to tendons because this vector stimulates few adverse tissue reactions. Basic fibroblast growth factor (bFGF) promotes collagen production in healing tendons. We transferred the exogenous bFGF gene to proliferating tenocytes by adeno-associated viral (AAV) vectors and investigated its effects on the expression of the collagen genes in an in vitro tenocyte model.

Methods

AAV2 vectors harboring the rat bFGF gene were constructed. Tenocytes were obtained from explant cultures of rat intrasynovial tendons and were distributed into 21 culture dishes and 8 wells. Tenocytes in 7 dishes were treated with AAV2 bFGF for 3 hours and then were cultured for 10 days. Tenocytes in 14 dishes (sham vector and nontreatment controls) did not receive the transgene. Efficiency of the gene transfer was evaluated by in situ β-galactosidase staining in 8 wells after treatment with AAV2 lacZ. Expression of the target genes was assessed by reverse-transcription polymerase chain reactions with primers specifically amplifying the target genes. Expression of bFGF and type I and III collagen genes was determined by quantitative analysis of the polymerase chain reaction products.

Results

Positive β-galactosidase staining confirmed the effectiveness of AAV2-mediated gene delivery to tenocytes. The level of expression of the bFGF gene was increased significantly after gene transfer. Levels of expression of type I and III collagen genes after transfer of the exogenous bFGF gene were increased significantly compared with those in the cells treated with sham vectors or in nontreatment controls.

Conclusions

Delivery of exogenous bFGF gene to tenocytes can increase significantly the levels of expression of the bFGF and type I and III collagen genes. AAV2 vectors provide a novel method for delivering growth factor genes to tenocytes. These findings warrant future in vivo study of the delivery of genes pertinent to tendon healing through AAV2-based gene therapy to enhance repairs of injured flexor tendons.

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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