The M2c Macrophages Promote Phalange Regenera-tion in An Organ Co-Culture with Amputated Mice Digit

Background: Delayed anti-inflammatory responses and subse-quent fibrotic scarring is the main causes for the fail to regrow of injured body parts such as limb/phalange in adult mammalians. Indeed, fully scar-free limb regeneration in adult salamanders, which can restore complete body parts, followed by appearance of anti-inflammatory macrophages in the site of injuries during the first 24 hours, after amputation. Consequently, modulation of inflammatory responses may provide valuable therapeutic approach for mammalians limb regeneration. Hence, this study aimed to analysis the local effects of IL-10 priming M2c mac-rophages on the digit tip regeneration of fetal mice forelimb in a co-cultured organ system, after amputation.Materials and Methods: To address this evaluation, we ob-tained hands of 18.5E-day-old C57BL/6J fetal mice, which amputations performed on digits. The mononuclear cells such as monocytes were obtained from C57BL/6J mice peripheral blood, cultured and enhances the phenotype of M2 macrophag-es subsequent induced by IL-10. Then, amputated hands co-cultured with M2c macrophage. In addition, some of samples treated with 40, 80 and 120 µg/ml concertation of IL-10 and TGF-β1cytokines as the main M2c macrophages secretions, re-spectively.Results: We found that wound closure occurred in all speci-men, three days’ post amputation (dpa). Increasing of the digit elongation achieved at 10 dpa in all groups that was associated with increased expression of specific markers of regeneration such as Msx1, Msx2 and Bmp4 (**P<0.01). In addition, en-hanced proliferation rate observed that is related with high level expression of Fgf-8, Ki67 (**P<0.01). Histological analyses in-dicated epidermal closure happened 3 dpa in all groups. H&E staining showed that apoptotic cell not identified in all groups. Surprisingly, full digit tip regenerated in co-cultured group. Indeed, Alizarin red-Alcian blue staining revealed new callus formed colored blue and rare bony-like tissue formation ob-served that colored red by alizarin red. Particularly, new tissue regrowth observed in 40 µg/ml IL-10 and 120 µg/ml TGF-β1 compared with control. In contrast, in control formed no re-markable digit elongation, as expected.Conclusion: We propose that existence of anti-inflammatory macrophages in injury site of digit can regenerate epithelial and mesenchymal tissues may actually be employed in limb regen-eration without scar formation in adult mammalian.

کلیدواژه ها:

TGF-β1 ، IL-10 ، Limb Regeneration ، M2c Mac-rophage ، Amputation