M۲c Macrophages enhance phalange regeneration of amputated mice digits in an organ co-culture system

Objective(s): Delayed anti-inflammatory responses and scar-formation are the main causes for inability of injured body parts such as phalanges to regrow in mammals. Salamanders can regenerate fully scar-free body structures, followed by the appearance of anti-inflammatory responses at the injured site immediately after amputation. This study aimed to evaluate the local regenerative effects of direct amplified anti-inflammatory signals on regeneration of amputated mice digit tips using M۲c-macrophages in a co-cultured organ system for the first time. Materials and Methods: We used the amputated digits from the paws of ۱۸.۵E day old C۵۷BL/۶J mice. Monocytes were obtained from peripheral blood and co-cultured with amputated digits, which subsequently enhanced the M۲c macrophage phenotype induced by IL-۱۰. We also examined the regenerative effects of IL-۱۰ and transcription growth factor-beta ۱ (TGF-β۱). Results: The regrowth of new tissue occurred ۱۰ days post-amputation in all groups. This regrowth was related to enhanced Msh homeobox-۱ (Msx۱), Msh homeobox-۲ (Msx۲), and bone morphogenic protein-۴ (Bmp۴) genes. Increased expression of fibroblast growth factor-۸ (Fgf-۸) also increased the proliferation rate. Histological analyses indicated that epidermal-closure occurred at ۳-dpa in all groups. We observed full digit tip regeneration in the co-cultured group. Particularly, there was new tissue regrowth observed with ۴۰ µg/ml of IL-۱۰ and ۱۲۰ µg/ml of TGF-β. In contrast, the control group had no remarkable digit elongation.Conclusion: We propose that a direct amplified anti-inflammatory response at the digit injury site can regenerate epithelial and mesenchymal tissues, and might be useful for limb regeneration without scar formation in adult mammals.

Keywords:

Anti-inflammatory - responses , IL-۱۰ , M۲c macrophage , Organ-culture , Paw regeneration , TGF-β