M2c Macrophages enhance phalange regeneration of amputated mice digits in an organ co-culture system

Document Type : Original Article


1 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 1665659911, Iran

2 Department of Developmental Biology, University of Science and Culture, Tehran, Iran

3 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR,Tehran, 1665659911, Iran


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 M2c-macrophages in a co-cultured organ system for the first time. 
Materials and Methods: We used the amputated digits from the paws of 18.5E day old C57BL/6J mice. Monocytes were obtained from peripheral blood and co-cultured with amputated digits, which subsequently enhanced the M2c macrophage phenotype induced by IL-10. We also examined the regenerative effects of IL-10 and transcription growth factor-beta 1 (TGF-β1). 
Results: The regrowth of new tissue occurred 10 days post-amputation in all groups. This regrowth was related to enhanced Msh homeobox-1 (Msx1), Msh homeobox-2 (Msx2), and bone morphogenic protein-4 (Bmp4) genes. Increased expression of fibroblast growth factor-8 (Fgf-8) also increased the proliferation rate. Histological analyses indicated that epidermal-closure occurred at 3-dpa in all groups. We observed full digit tip regeneration in the co-cultured group. Particularly, there was new tissue regrowth observed with 40 µg/ml of IL-10 and 120 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.


1. Jazwinska A, Sallin P. Regeneration versus scarring in vertebrate appendages and heart. J Pathol 2016; 238:233-246.
2. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: Molecular and cellular mechanisms. J Invest Dermatol 2007; 127:514-525.
3. Brancato SK, Albina JE. Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol 2011; 178:19-25.
4. Suzuki M, Yakushiji N, Nakada Y, Satoh A, Ide H, Tamura K. Limb regeneration in Xenopus laevis froglet. ScientificWorldJournal 2006; 6:26-37.
5. Stoick-Cooper CL, Moon RT, Weidinger G. Advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes Dev 2007; 21:1292-1315.
6. Christensen RN, Tassava RA. Apical epithelial cap morphology and fibronectin gene expression in regenerating axolotl limbs. Dev Dyn 2000; 217:216-224.
7. Vincent E, Villiard E, Sader F, Dhakal S, Kwok BH, Roy S. BMP signaling is essential for sustaining proximo-distal progression in regenerating axolotl limbs. Development 2020; 147.
8. Taghiyar L, Hesaraki M, Sayahpour FA, Satarian L, Hosseini S, Aghdami N, et al. Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice. J Biol Chem 2017; 292:10520-10533.
9. Han M, Yang X, Farrington JE, Muneoka K. Digit regeneration is regulated by Msx1 and BMP4 in fetal mice. Development 2003; 130:5123-5132.
10. Taghiyar L, Hosseini S, Safari F, Bagheri F, Fani N, Stoddart MJ, et al. New insight into functional limb regeneration: A to Z approaches. J Tissue Eng Regen Med 2018; 12:1925-1943.
11. Butterfield TA, Best TM, Merrick MA. The dual roles of neutrophils and macrophages in inflammation: A critical balance between tissue damage and repair. J Athl Train 2006; 41:457-465.
12. Mokarram N, Bellamkonda RV. A perspective on immunomodulation and tissue repair. Ann Biomed Eng 2014; 42:338-351.
13. Agata K, Saito Y, Nakajima E. Unifying principles of regeneration I: Epimorphosis versus morphallaxis. Dev Growth Differ 2007; 49:73-78.
14. Krzyszczyk P, Schloss R, Palmer A, Berthiaume F. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Front Physiol 2018; 9:419.
15. Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult salamander limb regeneration. Proc Natl Acad Sci U S A 2013; 110:9415-9420.
16. Stocum DL. Mechanisms of urodele limb regeneration. Regeneration (Oxf) 2017; 4:159-200.
17. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008; 214:199-210.
18. Rehermann B. Mature peritoneal macrophages take an avascular route into the injured liver and promote tissue repair. Hepatology 2017; 65:376-379.
19. Liu YC, Zou XB, Chai YF, Yao YM. Macrophage polarization in inflammatory diseases. Int J Biol Sci 2014; 10:520-529.
20. Shapouri-Moghaddam A, Mohammadian S, Vazini H, Taghadosi M, Esmaeili SA, Mardani F, et al. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 2018; 233:6425-6440.
21. Hao NB, Lu MH, Fan YH, Cao YL, Zhang ZR, Yang SM. Macrophages in tumor microenvironments and the progression of tumors. Clin Dev Immunol 2012; 2012:948098.
22. Ambrozova G, Martiskova H, Koudelka A, Ravekes T, Rudolph TK, Klinke A, et al. Nitro-oleic acid modulates classical and regulatory activation of macrophages and their involvement in pro-fibrotic responses. Free Radic Biol Med 2016; 90:252-260.
23. Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol 2010; 10:170-181.
24. Maumus M, Jorgensen C, Noel D. Mesenchymal stem cells in regenerative medicine applied to rheumatic diseases: role of secretome and exosomes. Biochimie 2013; 95:2229-2234.
25. Mescher AL. Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration. Regeneration (Oxf) 2017; 4:39-53.
26. Egawa S, Miura S, Yokoyama H, Endo T, Tamura K. Growth and differentiation of a long bone in limb development, repair and regeneration. Dev Growth Differ 2014; 56:410-424.
27. Frantz FW, Bettinger DA, Haynes JH, Johnson DE, Harvey KM, Dalton HP, et al. Biology of fetal repair: the presence of bacteria in fetal wounds induces an adult-like healing response. J Pediatr Surg 1993; 28:428-433; discussion 433-424.
28. Ozturk S, Deveci M, Sengezer M, Gunhan O. Results of artificial inflammation in scarless foetal wound healing: An experimental study in foetal lambs. Br J Plast Surg 2001; 54:47-52.
29. Soroosh P, Doherty TA, Duan W, Mehta AK, Choi H, Adams YF, et al. Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance. J Exp Med 2013; 210:775-788.
30. Ito T, Ito N, Saathoff M, Stampachiacchiere B, Bettermann A, Bulfone-Paus S, et al. Immunology of the human nail apparatus: the nail matrix is a site of relative immune privilege. J Invest Dermatol 2005; 125:1139-1148.
31. Ito T, Meyer KC, Ito N, Paus R. Immune privilege and the skin. Curr Dir Autoimmun 2008; 10:27-52.
32. Benhar I, London A, Schwartz M. The privileged immunity of immune privileged organs: the case of the eye. Front Immunol 2012; 3:296.
33. Carson MJ, Doose JM, Melchior B, Schmid CD, Ploix CC. CNS immune privilege: Hiding in plain sight. Immunol Rev 2006; 213:48-65.
34. Choi Y, Cox C, Lally K, Li Y. The strategy and method in modulating finger regeneration. Regen Med 2014; 9:231-242.
35. Saito M, Ohyama M, Amagai M. Exploring the biology of the nail: An intriguing but less-investigated skin appendage. J Dermatol Sci 2015; 79:187-193.
36. Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hill JA, Bassel-Duby R, et al. Macrophages are required for neonatal heart regeneration. J Clin Invest 2014; 124:1382-1392.
37. Duffield JS. Macrophages and immunologic inflammation of the kidney. Semin Nephrol 2010; 30:234-254.
38. Bain CC, Bravo-Blas A, Scott CL, Perdiguero EG, Geissmann F, Henri S, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol 2014; 15:929-937.
39. Munro DAD, Hughes J. The origins and functions of tissue-resident macrophages in kidney development. Front Physiol 2017; 8:837.
40. Al-Lamki RS, Bradley JR, Pober JS. Human organ culture: Updating the approach to bridge the gap from in vitro to in vivo in inflammation, cancer, and stem cell biology. Front Med (Lausanne) 2017; 4:148.
41. Yissachar N, Zhou Y, Ung L, Lai NY, Mohan JF, Ehrlicher A, et al. An intestinal organ culture system uncovers a role for the nervous system in microbe-immune crosstalk. Cell 2017; 168:1135-1148 e1112.
42. Smith EL, Kanczler JM, Oreffo RO. A new take on an old story: Chick limb organ culture for skeletal niche development and regenerative medicine evaluation. Eur Cell Mater 2013; 26:91-106.
43. Peroglio M, Gaspar D, Zeugolis DI, Alini M. Relevance of bioreactors and whole tissue cultures for the translation of new therapies to humans. J Orthop Res 2018; 36:10-21.
44. Sha Z, Wang L, Sun L, Chen Y, Zheng Y, Xin M, et al. Isolation and characterization of monocyte/macrophage from peripheral blood of half smooth tongue sole (Cynoglossus semilaevis). Fish Shellfish Immunol 2017; 65:256-266.
45. Seifert AW, Muneoka K. The blastema and epimorphic regeneration in mammals. Dev Biol 2018; 433:190-199.
46. Shieh SJ, Cheng TC. Regeneration and repair of human digits and limbs: fact and fiction. Regeneration (Oxf) 2015; 2:149-168.
47. Wermuth PJ, Jimenez SA. The significance of macrophage polarization subtypes for animal models of tissue fibrosis and human fibrotic diseases. Clin Transl Med 2015; 4:2.
48. Lenzo JC, Turner AL, Cook AD, Vlahos R, Anderson GP, Reynolds EC, et al. Control of macrophage lineage populations by CSF-1 receptor and GM-CSF in homeostasis and inflammation. Immunol Cell Biol 2012; 90:429-440.
49. Fernandez-Teran M, Ros MA. The apical ectodermal ridge: morphological aspects and signaling pathways. Int J Dev Biol 2008; 52:857-871.
50. Mori S, Sakakura E, Tsunekawa Y, Hagiwara M, Suzuki T, Eiraku M. Self-organized formation of developing appendages from murine pluripotent stem cells. Nat Commun 2019; 10:3802.
51. Lallemand Y, Bensoussan V, Cloment CS, Robert B. Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb. Dev Biol 2009; 331:189-198.
52. Ferrari D, Lichtler AC, Pan ZZ, Dealy CN, Upholt WB, Kosher RA. Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis. Dev Biol 1998; 197:12-24.