The capacity to regenerate complex tissue structures after amputation is limited to the digit tip in humans. In a comparable mammalian mouse model, the response is level-specific in that amputation at the terminal phalangeal element (P3) results in regeneration, but not at the next more proximal joint (P2). Recent isolation of skeletal cells from the P3 and P2 regions allow for comparative in vitro studies which can improve understanding of the processes that limit or drive regeneration. This dissertation will use regeneration-competent (P3) and – incompetent (P2) cells in in vitro model systems to further understand the initial stages of regeneration.

In the first study we sought to determine phenotypic differences in P2 and P3 cells in response to their physical microenvironment. We found that cytoskeletal modulation may play an important role in regeneration and that suspension culture may be a better in vitro model system to study regenerative processes than collagen gel culture, which is classically used for wound healing studies.

In the second study we investigated the use of oxygen concentration as a potential mechanism to alter the proliferation of P2 and P3 cells. Our results found that the proliferation rates of P2 and P3 cells are differently regulated by switch and level of oxygen concentration, respectively. Furthermore, we found that the cycle regulator p15 may be pivotal in the proliferative response of regeneration-incompetent P2 cells.

In the last study, we investigated the role of trophic factors secreted by fibroblasts and MSCs on P2 and P3 cells. We found that fibroblasts play a large role in regulating the proliferation and migration of P2 and P3 cells, critical processes in forming the stable cell mass required for in vivo regeneration. We have also shown that BMP signaling may be involved in the fibroblast-mediated changes in migration of P3 cells.

These studies used a variety of in vitro culture models to investigate factors that may be relevant for regeneration. Thus, the results of this dissertation can help advance the biological understanding of mammalian regeneration that will translate into treatment modalities for digit and limb amputation in humans.