Effect of interleukin-10 overexpression on the properties of healing tendon in a murine patellar tendon model

Abstract

Purpose: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine shown to inhibit scar formation in fetal wound healing. The role of IL-10 in adult tendon healing and scar formation, however, remains unknown. The objective of this study is to investigate the effect of IL-10 overexpression on the properties of adult healing tendon using a well-established murine model of tendon injury and a lentiviral-mediated method of IL-10 overexpression.

Methods: A murine model of patellar tendon injury was used and animals divided into 3 groups. Mice received bilateral patellar tendon injections with a lentiviral vector containing an IL-10 transgene (n = 34) or no transgene (n = 34). Control mice (n = 34) received injections of sterile saline. All animals then were subjected to bilateral, central patellar tendon injuries 2 days after injection and were killed at 5, 10, 21, and 42 days after injury. IL-10 content was analyzed by immunohistochemistry (n = 4/group). Tendon healing was evaluated by histology (n = 4/group) and biomechanical analysis (n = 10/group).

Results: Overexpression of IL-10 in patellar tendon was confirmed after injection of the lentiviral vector. IL-10 immunostaining was increased at day 10 in the IL-10 group relative to that in controls. Histologically, there was no significant difference in angular deviation between groups at day 21, but a trend toward decreased angular deviation in controls relative to that in empty vector group mice was seen at day 42. Biomechanically, the IL-10 group showed significantly increased maximum stress at day 42 relative to that in controls. Percent relaxation showed a trend toward an increase at day 10 and a significant increase at day 42 in the IL-10 group relative to that in controls.

Conclusions: This study demonstrates successful gene transfer of IL-10 into adult murine patellar tendon using a lentiviral vector. Although the effects of overexpression of IL-10 on adult tendon healing have not yet been fully elucidated, the current study may help to further clarify the mechanisms of tendon injury and repair.

Figure 1:

Expression of the eGFP reporter gene (eGFP), IL-10 transgene (IL-10 tran), and endogenous IL-10 (IL-10 endo) in adult mouse patellar tendon following injection of the lentiviral vector. Expression of the eGFP reporter gene and IL-10 transgene were found to peak at two days post-injection of the lentiviral vector. Gene expression was normalized to expression of the housekeeping gene, β-actin.

Figure 2:

Representative immunohistochemistry slides from day 10 post-injury demonstrate increased IL-10 immunostaining (brown staining) at the patellar tendon injury site in the IL-10 group (left slide) relative to saline controls (right slide).

Figure 3:

Maximum stress (MPa) at 10, 21, and 42 days post-injury. The empty vector group showed a trend toward increased maximum stress at day 10 post-injury relative to the saline controls, and maximum stress was significantly increased in the IL-10 group at day 42 post-injury relative to the saline controls. The asterisk (*) indicates a significant difference (p≤0.05) and the pound sign (#) denotes a trend (p≤0.1) when comparing between treatment groups within a post-injury time point.

Figure 4:

Percent relaxation (%) at 10, 21, and 42 days post-injury. Percent relaxation was significantly increased in the empty vector group relative to the saline controls at day 10 and 42 post-injury. It showed a trend toward an increase at day 10 and a significant increase at day 42 post-injury in the IL-10 group relative to the saline controls. The asterisk (*) indicates a significant difference (p≤0.05) and the pound sign (#) denotes a trend (p≤0.1) when comparing between treatment groups within a post-injury time point.

Figure 5:

Modulus (MPa) at 10, 21, and 42 days post-injury. No significant differences or trends were found in modulus between the three treatment groups at any of the post-injury time points.