Wound Healing Model

Written by Donald R Ferris

Axolotls are unique among adult vertebrates in their ability to regenerate amputated limbs. The first stage of regeneration involves fast wound healing followed by the formation of a blastema at the wound site and requires the presence of fibroblasts, keratinocytes and nerve. The wound epidermis is essential for blastema formation however little is known about molecular mechanisms involved. As a step towards understanding axolotl wound healing we have developed an in vitro skin model to study keratinocyte migration and gene expression. The model comprises a ‘donut’ shaped explant formed by taking a 6mm full thickness punch biopsy of axolotl (Ambystoma mexicanum) limb skin and creating a 2 mm central circular wound. The ‘donuts’ were placed on collagen gels and incubated in culture medium. Wound healing was examined by video microscopy and immunocytochemistry. Keratinocyte migration usually started within 2 hrs of plating the explants and proceeded rapidly (~300μm2/hr). The rate of cell migration was comparable to axolotl keratinocyte migration observed in vivo and an order of magnitude faster than mammalian keratinocyte migration. Re-epithelialization of the wound bed was normally complete within 10 hrs. Time lapse video microscopy revealed flattened lamellapodia extending from cells at the migrating wound edge. By 24 hrs the entire wound bed was covered by a multilayered epidermis. Although fibroblasts were present in the full thickness skin biopsy very few cells migrated into the collagen gel. The explants could be maintained in culture for at least 3 days. Keratinocyte migration was reversibly inhibited by matrix metalloproteinase (MMP) inhibitors and latrunculin A indicating that migration was dependent on MMP activity and actin polymerization. The ‘donut’ model provides simple novel system for generating wound epidermis and for studying axolotl wound healing. Understanding the mechanisms underlying wound healing in animals that regenerate is likely to lead to novel strategies for improving mammalian wound healing.