Regeneration of grade 3 ankle sprain, using the recombinant human amelogenin protein (rHAM+) in a rat model

Salem Hanhan, Koby Goren, Amit Rivkin, Faris Saba, Hani Nevo, Naama Dar, Dekel Shilo, Meir Liebergall, Oded Shoseyov, Dan Deutsch, Anat Blumenfeld, Amir Haze*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Lateral ligament tears, also known as high-grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+) induced regeneration of fully transected rat medial collateral ligament, a common proof-of-concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 μg/μL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 μg/μL rHAM+ was not as efficient as 0.5 μg/μL rHAM+. Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 μg/μL rHAM+, and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.

Original languageAmerican English
Pages (from-to)1540-1547
Number of pages8
JournalJournal of Orthopaedic Research
Issue number7
StatePublished - Jul 2021

Bibliographical note

Publisher Copyright:
© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC


  • amelogenin
  • ankle sprain
  • calcaneofibular ligament
  • rat model
  • regeneration


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