The Mexican axolotl (Ambystoma mexicanum (Shaw and Nodder, 1798)) is an urodele amphibian with intrinsic regenerative capacity in a range of organs and tissues e.g. heart, spinal cord and appendages.
Urodeles represents one of few tetrapods that can recreate the embryonic/undifferentiated environment of cells to regenerate once differentiation has already occurred. When trauma occurs, the wound is sealed off by wound epithelium which stems from migrating epithelial cells. Dedifferentiated local cells are activated in the stump accumulate at the tip and form the blastema. The blastema cells proliferate and control the pattern formation so the limb reforms in the right way. This includes the position of cartilage cells, and it takes around 2-3 months to have a fully differentiated limb. Most studies although only look at the first stages of the regenerated limb (until cartilage formation) whereas calcification may take even longer.
The route of calcium uptake during limb regeneration in the axolotl is not known. However, there is anecdotal evidence for the observation that age matched axolotls develop mineralized limbs at different rates in different laboratories with varying water supply and food, suggesting an effect of the of ambient calcium level.
Our hypothesis is that calcium uptake during limb regeneration is maintained by ambient available calcium dissolved in either the food or aquatic environment, and low levels of ambient calcium results in lack of mineralization in the regenerated appendage and/or results in transient/permanent osteopenia in the remaining skeleton.
Materials and methods
Axolotls (n=24) is divided into four groups respectively: Control (n=6), calcium enriched food (n=6), Calcium free water and enriched food (n=6), High calcium water (0,61 g/l CaCl2) and food(n=6). Calcium enriched pellets was soaked in a saturated CaCl2 solution (850g/l) for 1h and dried for 24h at 21oC.
To assess mineral density the animals was scanned by non-invase microCT prior to amputation. It is planned to be done again 3 months post amputation, 4,5 months and before euthanization.
Bone mineral content will subsequently be validated by ashing and some animals will sacrificed at different stages of mineralization for histological staining, but this has yet to be done.
Results:
To ascertain the amount of calcium uptake trough eating we found the calcium enriched pellets to have mean factor of 4.67 times more calcium in them than the normal pellets (Ca enriched mean: 56ppm and normal pellets mean: 12ppm).
Conclusion: With the initial assessment of regeneration between the different groups. We expect to see a difference in mineralization.
Experimental group setup (A). CaCl2 enriched pellets were made by soaking pellets into a saturated CaCl2 solution for 1h and dried for 24h at 21C. (1) Control. (2) 40% Holtfreters, fed enriched pellets (3) The solution was made to be isosmotic without CaCl2. (4) The solution was made to be isosmotic but with 0.61g/l CaCl2. Figure Created with BioRender.com. CaCl2 levels in food pellets (B). Mean levels of calcium in food pellets soaked in dem water (n=10) and CaCl2 enriched (850g/l) (n=10).
