Microcomputed tomography staging of bone histolysis in the regenerating mouse digit.

Humans and mice have the ability to regenerate the distal digit tip, the terminal phalanx (P3) in response to amputation. What distinguishes P3 regeneration from regenerative failure is formation of the blastema, a proliferative structure that undergoes morphogenesis to regenerate the amputated tissues. P3 regeneration is characterised by the phases of inflammation, tissue histolysis and expansive bone degradation with simultaneous blastema formation, wound closure and finally blastemal differentiation to restore the amputated structures. While each regenerating digit faithfully progresses through all phases of regeneration, phase progression has traditionally been delineated by time, that is, days postamputation (DPA), yet there is widespread variability in the timing of the individual phases. To diminish variability between digits during tissue histolysis and blastema formation, we have established an in-vivo method using microcomputed tomography (micro CT) scanning to identify five distinct stages of the early regeneration response based on anatomical changes of the digit stump. We report that categorising the initial phases of digit regeneration by stage rather than time greatly diminishes the variability between digits with respect to changes in bone volume and length. Also, stages correlate with the levels of cell proliferation, osteoclast recruitment and osteoprogenitor cell recruitment. Importantly, micro CT staging provides a means to estimate open versus closed digit wounds. We demonstrate two spatially distinct and stage specific bone repair/regeneration responses that occur during P3 regeneration. Collectively, these studies showcase the utility of micro CT imaging to infer the composition of radiolucent soft tissues during P3 blastema formation. Specifically, the staging system identifies the onset of cell proliferation, osteoclastogenesis, osteoprogenitor recruitment, the spatial initiation of de novo bone formation and epidermal closure.

Aging Delays Epimorphic Regeneration in Mice.

Epimorphic regeneration is a multitissue regeneration process where amputation does not lead to scarring, but blastema formation and patterned morphogenesis for which cell plasticity and concerted cell-cell interactions are pivotal. Tissue regeneration declines with aging, yet if and how aging impairs epimorphic regeneration is unknown. Here, we show for the first time that aging derails the spatiotemporal regulation of epimorphic regeneration in mammals, first, by exacerbating tissue histolysis and delaying wound closure, and second, by impairing blastema differentiation and skeletal regrowth. Surprisingly, aging did not limit stem cell availability in the blastema but reduced osteoblast-dependent bone formation. Our data suggest that aging delays regeneration not by stem cell exhaustion, but functional defects of differentiated cells that may be driven by an aged wound environment and alterations in the spatiotemporal regulation of regeneration events. Our findings emphasize the importance of accurate timing of signaling events for regeneration and highlight the need for carefully timed interventions in regenerative medicine.