A Study Of Expression Of Growth Factors In The Early Tendon Healing Period And Delivery Of Transgene For Tendon Repairs

Objective:(1) To observe the discipline of mRNA experssion level of CTGF, TGF-β, IGF-1, VEGF, PDGF-B and bFGF during healing early periods of injured flexor tendons, and the protein production expression action of TGF-β, bFGFand IGF-1 at multiple time-points during the early healing period in a chicken model.(2) To investigate efficiency and distribution of gene delivery to the injured tendons and tissue reactions caused by different vectors(3) To investigate efficiency of gene delivery of the adenoviral, adeno-associated viral and liposome-plasmid vectors to the uninjured tendons, and to explore the bFGF gene expression by AAV2 vectors during tendon healing.(4) To investigate the in vitro and in vivo effects of delivery of microRNA (miRNA) to silence expression of the transforming growth factorβ1 (TGFβ1) genes and its influence on expression of the typeⅠ,Ⅲcollagen and connective tissue growth factor (CTGF) genes in cultured tenocytes and injured tendons.Method:(1) Seventy-four long toes of 37 white Leghorn chickens were used. The flexor digitorum profundus tendons of 60 toes were surgically repaired after complete transection and were harvested for analysis on days 3, 5, 7, 9, 14, or 21 post-surgery. Expression of 6 growth factors at 4 post-surgical time-points was studied with real-time quantitative polymerase chain reactions, and production and distribution of 3 growth factors at all 6 time-points were studied by immunohistochemical staining with antibodies. Fourteen tendons that underwent no surgery served as day 0 controls. Tendon healing status was also assessed histologically.(2) Using a microinjection technique, 10μl of pCMV-EGFP, pCAGGS-EGFP, AAV2-EGFP, and Ad5-EGFP, harboring enhanced green fluorescence protein (EGFP) gene, respectively, were injected to two sites of the proximal stump of 48 transected digital flexor tendons in 18 chickens. At 3, 7, 14, and 21 days, the tendons were harvested. Under a fluorescence microscope, distribution and expression of EGFP was examined using frozen tissue sections. The tendon sections were also stained with hematoxylin and eosin to examine inflammation caused by these vectors. 24 flexor tendons were not injured, serving as the controls.(3) pCMV-EGFP, AAV2-EGFP, and Ad5-EGFP were injected to the uninjured digital flexor tendons of the chickens. At 3, 7, 14, and 21 days, the tendons were harvested. Under a fluorescence microscope, the expression of EGFP was examined. The AAV2-bFGF was injected to the injured digital flexor tendons of the chickens. Immunohistochemical staining of the tendons treated with AAV2-bFGF and non-treated with AAV2-bFGF was carried out at 2, 3 and 4 weeks after surgery.(4) Four miRNAs TGFβ1 DNA and one sham sequence vector control were designed and synthesized according to chicken TGFβ1 mRNA sequence. They were used to produce 5 plasmid expression vectors (miRNA #1,#2,#3,#4 and negative control). Cultured chicken tenocytes were transfected with these vectors. Under a fluorescence microscope and flow cytometry, the expression of EGFP was examined, to evaluate the transfection efficiency of the vectors. Expression of TGFβ1, collagen I, III, and CTGF genes were measured using real-time PCR. miRNA #1 plasmid, which had the highest inhibitory effects to the TGFβ1 gene was injected into injured digital flexor tendons of chickens. At week 1 and 6 postsurgery, expression of TGFβ1, collagen I, III, and CTGF gene were measured using real-time PCR.Results:(1) Throughout the early tendon healing period, connective tissue growth factor (CTGF) and transforming growth factor-beta (TGF-β) showed high levels of gene expression. Levels of gene expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) were high or moderately high. Expression of the TGF-βgene was up-regulated after injury, while basic fibroblast growth factor (bFGF) gene was down-regulated at all post-surgical time-points and expressed at the lowest levels among 6 growth factor genes 2 to 3 weeks post-surgery. Platelet-derived growth factor B (PDGF-B) gene was also minimally expressed. Findings of Immunohistochemistry corresponded to TGF-β, bFGF, and IGF-1 gene expression. (2) Compared with normal tendon tissues, the EGFP expression was observed in tendons at 3, 7, 14 and 21 day post-injection. The EGFP expression was observed at 3 days, and was the highest at 7 days for all vectors. At 14 days, we observed a decrease in the EGFP expression. EGFP was distributed even in the injected tendon segment adjacent to the cut level. The EGFP expression in the tendons injected with AAV2-EGFP and Ad5-EGFP was higher than that with pCMV-EGFP and pCAGGS-EGFP injection. We did not find remarkable differences in the EGFP expression between AAV2-EGFP and Ad5-EGFP vectors. Tissue reactions of the tendons caused by the liposome–plasmid vector (including pCMV-EGFP and pCAGGS-EGFP) were the most prominent among all vectors. Infiltration of Inflammatory cells, chiefly lymphocytes and neutrophilic granulocytes, were observed. Inflammatory reactions in the tendons injected with AAV2 vectors were the least severe.(3) Compared with normal tendon tissues, the EGFP expression was observed in tendons at 3, 7, 14 and 21 day post-injection. The EGFP expression was observed at 3 days, and was the highest at 7 days for all vectors. At 14 days, we observed a decrease in the EGFP expression. The EGFP expression in the tendons injected with AAV2-EGFP and Ad5-EGFP was higher than those of pCMV-EGFP injection. We did not find remarkable differences in the EGFP expression between AAV2-EGFP and Ad5-EGFP vectors. The AAV2-bFGF treatment remarkably increased the expression of bFGF at 2, 3 and 4 weeks after surgery.(4) EGFP expression was confirmed in tendon cell using fluorescence microscope and flow cytometry which showed that 20~25% of the tenocytes were transfected by the miRNA vectors. Compared with the negative control, expression of the TGFβ1gene in the tenocytes treated with miRNA #1 and #2 was decreased by 68% and 43%, respectively. In the cells treated with miRNA #1, expression of the typeⅢcollagen and CTGF genes was decreased by 70% and 68%, respectively. These changes were statistically significant. At week 1 postsurgery, expression of the TGFβ1 gene in the injured digital flexor tendons decreased by 67%, but expression of the collagen I, III, and CTGF genes were not changed. At week 6, expression of the TGFβ1 and typeⅢcollagen genes was decreased by 56% and 58%, respectively; the changes were statistically significantConclusion: (1) In this model, up to post-surgical 3 weeks, gene expression and production of TGF-βare high and are up-regulated in this healing period. However, expression of the bFGF gene and protein is low and decreases in the healing tendon. CTGF, VEGF, and IGF-1 genes are expressed at high or moderately high levels, but PDGF-B is minimally expressed.(2) Microinjection to two sites of each tendon stump delivers the transgene to the entire tendon segment adjacent to the cut. Efficiency of gene delivery by the AAV2 and Ad5 vectors is the highest among 4 vectors tested. Expression levels peak at 7 days post-injection. AAV2 vector causes the slightest tissue reactions in the tendons. The study suggests that the AAV2 vector is a promising gene delivery vector and microinjection is practical for tendon gene therapy.(3) Efficiency of gene delivery by the AAV2 and Ad5 vectors is the higher than the liposome-plasmid vectors. The tendons with AAV2-bFGF treating express the bFGF strongly, which can be maintained at a high level up to 4 weeks. The study suggests that the AAV2 and adenoviral vectors are promising gene delivery vectors for tendon gene therapy.(4) Expression of the TGFβ1, collagen III, and CTGF genes decreased substantially in tendon cells treated with miRNAs. Expression of the TGFβ1 gene was reduced in injured tendons at week 1 postsurgery; expression of both the TGFβ1 and collagen III genes was decreased at week 6. The study indicates that delivery of miRNA to silence expression of the TGFβ1 gene may be a promising in reducing tendon adhesion formations.