Regenerative Properties in Mammals

T here are many ways to enhance wound healing and repair in mammals. However, mammals do not have the ability to regenerate limbs.^1,2 That is, tissue will not grow in a new direction and begin to form new tissue, let alone completely form a functional organ. Regeneration is often seen in amphibians. There are examples of this occurrence in mammals. Every year, male deer grow antlers that fall off and then re-grow,^3–5 and human fingertips have been shown to regenerate.^6–9 An interesting discovery surfaced many years ago. If a hole is created through the cartilage of a rabbit ear, the hole will close.^10–12 Replicating this event in the mouse would be of particular interest, since the mouse, a standard animal used in laboratory study, provides a wealth of genetic information, inbred strains, reagents, and mutants, such as single gene knockout mice. However, mice do not close through-and-through ear holes.¹³ Instead, the area heals through the usual wound repair mechanisms, including sealing off of the wound, a provisional matrix, and fibrosis. The exception to this is the MRL mouse, which has been used to study autoimmune disease for the last 40 years.^14–16 The MRL mouse closes a through-and-through ear hole wound.^17–21 The mechanisms by which this mouse heals such a wound could reveal how a mammal may regenerate a limb, though future research is warranted. Remodeling the Mammalian Wound After an injury, reepithelialization is a primary event that covers the wound site whether repair or regeneration occurs. In non-mammalian species that regenerate tissue, the regeneration site shows rapid reepithelialization of the wound. The reepithelialization process, which can be complete in less than six hours, involves both migration and proliferation of cells. Reepithelialization takes place within 48 hours in the MRL mouse ear wound compared to 5–10 days in a control (C57BL/6) mouse.¹⁷ It is not clear how this might facilitate a regenerative response, but current research on this model is focused on early events that differentiate MRL from B6 ear hole healing. Another important difference is the formation of a basement membrane between the epithelial and dermal tissue layers during healing. It appears that a key process in the ongoing regenerative response is the cross communication between the epidermal and mesenchymal cells in the regenerate. The basement membrane acts as a barrier to such molecular interactions. It has been observed that there is no basement membrane between the epidermis and the dermal layer and assumed that important molecular interactions are occurring in the amphibian regenerating limb.^22,23 It has been shown that if the formation of a basement membrane can be induced, this will stop the regeneration process completely and induce scar formation.²⁴ In the MRL and B6 ear wound, a basement membrane similar to a normal mammalian wound healing response forms.²⁵ However, by Day 5 post-injury, a dramatic difference is apparent between the healer and nonhealer ear. The MRL basement membrane disappears. This change can be visualized using dye-stained tissue and epifluorescence. Soon after this event, MRL blastema growth is evident. Most likely, this remodeling response is due to proteases that are present in the wound. Certainly, protease-mediated remodeling is an important event in wound repair,^26–29 and matrix metalloproteinase (MMP) molecules play a central role in this process. In the MRL/B6 injury model, the author and colleagues have shown that neutrophils that are positive by immunohistochemistry for MMP-2 and -9 and tissue inhibitor of metalloproteinase (TIMP)-2 and -3 enter the wound site as early as Day 1 post-injury.²⁵ This activity peaks in neutrophils at approximately Day 3. Macrophages also are positive for MMPs and TIMPs and peak at Day 5. In the MRL ear wound, MMPs are higher (both by zymography and number of positive cells), and TIMPs are lower (by cell number and Western blot analysis) than levels seen in the B6 ear wound.²⁵ This type of MMP response is similar to responses seen in many regenerating non-mammalian models, though in these cases, the epithelial and mesenchymal tissues express these molecules.^30–34 The significance of MMP expression in MRL ear wounds has been shown by specifically blocking MMP activity, leading to reduced MRL ear hole closure (preliminary results, data not shown). Discussion The aforementioned events lead to the growth of the blastema. The closure of the MRL ear hole wound brings with it cellular condensations, differentiation, and the formation of new cartilage and muscle in the ear. Once the process of blastema growth starts, it may be possible that the process of limb regeneration proceeds by itself as a process that recapitulates development. It should be pointed out that the MRL does not grow back digits, though the blastema forms (data not shown). Studies using cortical brain injuries in the MRL show a healing response that differs from the control mouse but only shows such differences during the first four days after injury. This corresponds to a MMP response at the injury site that starts but cannot be maintained.³⁵ Another issue is the need for an inflammatory response. The inflammatory response is activated in response to an injury in general. This includes the early migration of neutrophils into the wound site (within 24 hours) followed by macrophages and various populations of lymphocytes. As discussed in relationship to the MMP response, the inflammatory response is greater in the MRL ear injury. In addition to the MMPs and TIMPs, the inflammatory cellular response brings in many molecules that activate important processes, such as remodeling and neovascularization. On the other hand, it is believed that the lack of an inflammatory response during fetal scarless healing is responsible for the lack of scar formation. It has also been shown that inflammatory molecules are down regulated in the MRL ear wound compared to a control.³⁶ This is an issue that needs to be resolved. Conclusion Comparing the MRL mouse ear hole injury model to a control mouse injury provides useful information to further understand how a blastema is formed, what processes are involved in cell growth and differentiation, and how this research can be applied to mammalian regeneration.