Tendon healing in vitro: bFGF gene transfer to tenocytes by adeno-associated viral vectors promotes expression of collagen genes.
OBJECTIVE
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 beta-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 beta-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.
Wang
Xiao Tian
XT
Department of Surgery, Roger Williams Medical Center, Boston University School of Medicine, Providence, Rhode Island, USA.
Liu
Paul Y
PY
Xin
Ke-Qin
KQ
Tang
Jin Bo
JB
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013710:Tendons / Q000166:cytology*; D014161:Transduction, Genetic; D014945:Wound Healing / Q000235:genetics
D024042:Collagen Type I; D024061:Collagen Type III; D016222:Fibroblast Growth Factor 2