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the casting off of the distal bone fragment. Sequential micro-computed tomography
(μCT) 3-D renderings illustrate the intense degradation of the bone stump in
response to amputation , and showcases the eventual expelling of the distal bone
stump evident by 10 days post-amputation (DPA; Fig. 5.1i ). In summary, the early
stages of this amputation model is characterized by a slow and variable wound heal-
ing response and an extensive histolytic response of bone tissue that results in an
injury-induced re- amputation of the digit [ 6 ]. This re- amputation causes a decrease
in both bone volume and bone length that is signifi cantly greater than that caused
by the initial amputation injury (Fig. 5.1g, h ).
The timing of wound closure and blastema formation is tightly linked; the blas-tema forms rapidly once wound closure is complete. The blastema consists of a
population of undifferentiated mesenchymal cells with a relatively high prolifera-
tion index that is proximally bounded by the bone stump and distally bound by the
thickened wound epidermis (Fig. 5.1j ). One consequence of the injury-induced re-
amputation is that the distal wound site where blastema formation occurs is directly
adjacent to the P3 marrow region which is highly vascularized. We note that the
transition between the cell dense blastema and the highly vascularized but relatively
less cell dense bone marrow is quite dramatic (Fig. 5.1j ). The next step of regenera-
tion is the redifferentiation of the blastema , which occurs via intramembranous ossi-
fi cation , with no evidence of chondrogenesis [ 6 , 8 , 9 ]. The bone redifferentiation
stage begins at approximately 12 DPA, with initial boney condensations showing
continuity with the bone stump at both the proximal boundary of the blastema and
the dorsal periosteal surface (Fig. 5.1j ). The overt intramembranous redifferentia-
tion of the blastema occurs in a proximal to distal fashion, resulting in an increase
in bone and associated surrounding connective tissue length, as well as an increase
in bone volume (Fig. 5.1h, k, l ). By 28 DPA, the digit has completed regeneration,
including integration of the newly formed bone with the bone stump, reconstitution
of the marrow cavity, and regeneration of the surrounding connective tissues, i.e.
vasculature, dermis, epidermis, and nail. The digit regenerates to the pre-amputation
bone length and characteristic pointed edge, but notably, the resulting regenerate
exhibits a disorganized trabecular bone pattern and an overshoot in bone volume
(Fig. 5.1l ). By 128 DPA, the trabecular bone of the regenerate has condensed, yet
the relative disorganized morphology is easily distinguishable from the original
bone stump , thus the regeneration response does not result in a perfect replica of the
amputated structure (Fig. 5.1m ).
While digit tip regeneration in adult mice digit embodies a rather complex butcoordinated series of events leading to blastema formation and re-differentiation,
the events associated with neonatal and fetal regeneration appear to be less com-
plex. Neonatal digits are structurally patterned but immature, containing cells still
undergoing chondrogenesis and ossifi cation is just initiating [ 8 ]. Unlike adult digit
amputations , the neonatal wound epidermis closes directly over the stump bone,
however the timing to completely close the wound is highly variable by comparison
to non-regenerative amputations [ 10 ]. The osteoclast-mediated bone degradation
response observed in adult amputations is absent in neonates, and regenerative out-
growth is not preceded by an injury-induced re-amputation of the stump. Once
L.A. Dawson et al.