Growth differentiation factor-5 regulation of extracellular matrix gene expression in murine tendon fibroblasts

Abstract

The synthesis and organization of extracellular matrix (ECM) of tendon, in resting and states of repair, are governed by fibroblasts. Growth differentiation factor-5 (GDF-5) may enhance the cellular response to tendon injury, thus improving the structural outcome of the regenerative tissue. This study was an attempt to identify potential mechanisms controlling the response of fibroblasts to injury and GDF-5, in the pursuit of improved tissue regeneration. There were two sets of experiments. Isolated mice Achilles tendon fibroblasts were treated with different concentrations of rGDF-5 (0-100 ng/ml) for 0-12 days in cell culture. The temporal effect of rGDF-5 on ECM gene expression was analysed for type I collagen and aggrecan expression. Microarray and gene expression analysis were performed on cells treated with 100 ng/ml for 4 days. Forty-five mice underwent bilateral mid-substance Achilles tendon tenotomy and suture repair. Repair sites were injected with 10 µg rGDF-5 or saline. Tendons were assessed histologically at 2, 4 and 6 weeks. Expression of ECM genes procollagen IX, aggrecan, matrix metalloproteinase 9 and fibromodulin were upregulated. Proinflammatory reaction genes were downregulated. rGDF-5 led to an increase in total DNA, glycosaminoglycan (GAG) and hydroxyproline (OHP). The OHP:DNA ratio of fibroblast cultures was increased over all time points, with increased GAG:DNA at day 12. rGDF-5 treatment showed improved collagen organization over controls. The results delineate the mode of action of rGDF-5 at the cellular and gene level. rGDF-5 could play a role in tendon repair and be used for future therapies that promote tendon healing.