618. bFGF or VEGF Gene Transfer Via AAV2 Vectors To the Tendon Activates Multiple Genes Critical To Tendon Growth and Regeneration: An In Vivo Study

Abstract

Purpose: Tendon healing is innately weak and transfer of growth factor gene therapy may activate multiple genes necessary or specific for tendon growth and regeneration. In this study we transferred bFGF or VEGF gene by AAV2 vectors to the healing tendon in a chicken flexor tendon model. We investigated how endogenous tendon healing related genes were activated after the delivery of AAV2-bFGF or AAV2-VEGF and the differences between epitenon and endotenon using a laser capture microdissection ((LCM).Methods: Eighteen digital flexor tendons from the big toes of 9 chickens were used. The tendons were completely transected and divided into three groups: AAV-bFGF injection, AAV-VEGF injection and no-injection control. After AAV injection, the tendons were surgically repaired. At 4 weeks after surgery, the tendons were harvested. The formalin-fixed, paraffin-embedded tendons were sectioned. LCM was used to dissect epitenon and endotenon separately. The tissue was subsequently analyzed for gene expression of collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), proliferating cell nuclear antigen (PCNA), fibronectin 1 (FN1), scleraxis (SCX) and tenascin C (TNC) using RT-qPCR analysis.Results: In AAV-VEGF and AAV-bFGF injected tendons, we found significant increases in expression of FN1, PCNA, SCX, COL1A1 and COL3A1 genes (p < 0.05 or p<0.01) compared with no-injection control. In the AAV-bFGF injected tendon, SCX was expressed 5-fold higher in the endotenon than in the epitenon. The increases in TNC gene expression did not reach statistical significance, but interestingly, TNC gene was expressed highly in the endotenon (10-fold greater than in the epitenon) in AAV-VEGF injected tendon. The TNC in the endotenon of AAV-VEGF-injected tendon was raised by 55-fold compared with that of the non-injected tendon.Discussion: Our results demonstrated a drastic increase in expression of studied genes specific to tendon growth and regeneration after AAV2-bFGF or AAV2-VEGF therapy. We also found precisely the epitenon and endotenon are equally responsive to the gene therapy. This study provides evidence at genetic levels to support the effective changes of the gene expression by the gene therapy, which may be key mechanistic findings to support the use of these gene therapies.