Amphibians as research models for regenerative medicine

Abstract

The ability to regenerate bone across a critical size defect would be a marked clinical advance over current methods for dealing with such structural gaps. Here, we briefly review the development of limb bones and the mandible, the regeneration of urodele limbs after amputation, and present evidence that urodele and anuran amphibians represent a valuable research model for the study of segment defect regeneration in both limb bones and mandible.

Keywords: critical size defect; endochondral bone; frog; growth factors; intramembranous bone; limb regeneration; salamander; scaffolds.

Figure 1

(A) Longitudinal section through a young axolotl forelimb four days after amputation through the distal end of the radius (R) and ulna (U). An accumulation blastema has formed under the wound epithelium, which is thickening into an apical epidermal cap (red arrowhead). (B) By 21 days, the blastema has grown and differentiated into the missing limb parts. Line indicates the amputation level. Hematoxylin and eosin stain.

Figure 2

Methylene blue-stained whole mounts of (A) control and (B) RA-treated (100 µg/gm body wt) young axolotls amputated through the distal radius and ulna. The line indicates the amputation level. The control regenerated a normal hand, whereas the RA-treated animal regenerated a serially duplicated limb. In (B), the arrow points to the shoulder girdle, and h indicates the humerus of the serially duplicated limb.

Figure 3

Radiograms of control (A, B, A′ and B′) and RA-treated (C, D, C′ and D′) axolotls limbs amputated through the mid-radius (R) and ulna, (U) 45 days (A–D) and 73 days (A′–D′) after amputation (dpa). The axolotls were large juveniles and thus took much longer to regenerate than the animals in Figures 1 and 2. The RA was delivered in a PLGA scaffold implanted into a dorsal fin tunnel. The radiopacity of the regenerating bone is less than the pre-existing bone. At a dose of 10 µg/gm body wt, two small skeletal protrusions (SP) were formed at the junction of the pre-existing and regenerated ulna bone, while at a dose of 20 µg/gm body wt a single skeletal protrusion was formed.

Figure 4

Three-dimensional CT tomographs of skeletal protrusions in limbs treated with 10 µg/gm body wt. and harvested at two months postamputation. The images of (A–C) are the same anatomical region captured from different angles of view. The curved arrows show the rotation of (A) to give the view in (B), and the rotation of (B) to give the view of (C). Arrowheads indicate openings into the skeletal protrusions (Marrow cavity 1 and 2). R = radius, U = ulna, D = dorsal, V = ventral.