Adult mouse digit amputation and regeneration: A simple model to investigate mammalian blastema formation and intramembranous ossification

Lindsay A. Dawson*, Regina Brunauer, Katherine N. Zimmel, Osama Qureshi, Alyssa R. Falck, Patrick Kim, Connor P. Dolan, Ling Yu, Yu Lieh Lin, Benjamin Daniel, Mingquan Yan, Ken Muneoka

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (μCT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo μCT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Original languageEnglish
Article numbere59749
JournalJournal of Visualized Experiments
Issue number149
StatePublished - Jul 2019
Externally publishedYes


  • Amputation
  • Blastema
  • Decalcification
  • Developmental Biology
  • Digit
  • Epimorphic regeneration
  • Fluorescence immunohistochemistry
  • Intramembranous ossification
  • Issue 149
  • Microcomputed tomography
  • Osteoprogenitors


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