This dissertation is composed of three sections each identifying integral biological events associated with improved ligament healing while using allogeneic MSCs. The first section compares two doses of MSCs to determine key cellular and cytokine expression levels that result in stronger ligaments, the second part builds on these findings by enhancing allogeneic MSC therapy through cell priming, and the third section defines local and systemic cytokine patterns that characterize an improved ligament healing environment from an inflammatory perspective.

In the first section, two different cell doses (low dose 1x106 and high dose 4x106 MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. The lower dose of MSCs resulted in improved ligament mechanics at day 14 and showed an improved cellular profile with fewer pro-inflammatory cells and cytokines compared to the high dose of MSCs. This study outlined cellular responses that correlated with improved ligament functional properties as well as demonstrated the potential of allogeneic MSCs (higher dose) to create a more inflammatory healing environment illustrating the importance of cell dosage.

The second section examined the role of priming cells with a pro-inflammatory cytokine (polyinosinic and polycytidylic acid) prior to administration into an injured ligament. Healing was studied at days 4 and 14 post-injury in ligaments that received unprimed MSCs (1x106 cells), primed MSCs (1x106 cells), and controls (no MSCs). Priming cells yielded stronger ligaments compared to the unprimed MSC group at day 14 along with increased type 2 macrophage infiltration and procollagen 1α deposition at day 4 of healing. Both primed and unprimed MSCs were detected in the healing region 14 days post-application, however, significantly fewer remained in the primed group. There was a pattern of co-localization of MSCs with both endothelial cells and pericytes in the healing region suggesting a strong interaction and role in angiogenesis for both primed and unprimed MSCs.

The third section measured cytokine fluctuations with the goal of identifying critical patterns contributing to the improved outcome from the use of primed MSCs. This analysis examined local and systemic changes in cytokine expression upon primed and unprimed MSC application. Systemic changes were monitored days 1-4 post-injury via serum collection. Local changes were measured at day 4 post-injury within the ligament homogenate. The primed cells resulted in a general dampening of several pro-inflammatory cytokines including GM-CSF, IL-6, TNFα, and IL-12 during the first few days after injury. However, there was also a general dampening of anti-inflammatory cytokine IL-10 in this group. Locally, the primed cells lead to increased antiinflammatory IL-1Ra within the healing matrix and decreased pro-inflammatory IL-1α. Summarizing the results, priming MSCs appeared to alter both the systemic and local healing environments by reducing inflammation leading to an overall improvement in ligament healing