95infl ammation is induced by RANK Ligand (RANKL) stimulation [ 49 ]. Both RANK
and RANKL are cell surface receptors and this signaling pathway is down- regu-
lated by Osteoprotegerin (OPG) , a secreted decoy receptor for RANK [ 50 ].
Regulation of osteoclastogenesis by this pathway is regulated by oxygen sensing
mechanism in osteoblasts by responding to hypoxia by enhancing Hypoxia Inducing
Factor 2a activity which directly up-regulates expression of OPG [ 51 ]. In this way
hypoxia induces the termination of an osteoclastogenic response. This model pro-
vides a general mechanism in which a blastema hypoxic event can trigger osteo-
blasts within the blastema to produce OPG thereby inhibiting osteoclast activity
and signaling the transition from a degradative phase to an anabolic phase of digit
regeneration.
51510 20 25 30ControlDermabondUADays post amputationPercent amputation bone volume (^00)
20
40
60
80
100
120
140
160
(^071014)
0.0005
0.001
0.0015
0.002
DPA
Low NOc/BPm
Norm
High
0
DPA10
Percent total are
DPA12DPA14
20
40
60
80
A D 100
F
K
GH
BC E
L
Fig. 5.6 P3 regeneration represents a dynamic oxygen microenvironment. ( a – d ) Oxygen pseudo-
shading of in vivo Hypoxyprobe‐1 Plus staining (<1.3 % oxygen) indicates hypoxic microenviron-
ments (<1.3 % oxygen) at ( a ) 10 DPA ( shading encircling the bone stumps distally), ( b ) 12 DPA
( shading in blastema; star emphasizing expanded area), and 14 DPA (isolated regions adjacent to
newly forming bone). ( d ) Quantifi cation of Hypoxybrobe-1 as a percentage of total area. ( e ) Effect
of daily HBO application on osteoclast numbers at 7, 10, and 14 DPA. Gray bars indicate HBO
treated digits ; black bars are controls. Results are expressed as mean ±SEM. Star indicates signifi -
cance. NOc/BPm: number of osteocalsts/bone perimeter. ( f ) Cyanoacrylic wound dressing
( Dermabond ) application results in a hypoxic epidermis. Arrow and arrowhead indicate
Hypoxyprobe-1 staining in both the dorsal and ventral wound epidermis, respectively. ( g )
Dermabond-treated digits exhibit early wound closure distal to the amputation plane ( asterisk ). ( h )
A representative Dermabond -treated digit illustrating wound closure and blastema formation
( asterisk ) by 6 DPA. ( i and j ) Immunostaining for the osteoclast marker Cathepsin K (CathK)
shows decreased osteoclast fusion ( arrow ) in Dermabond-treated digits and large multinuclear
immunopositive osteoclasts ( arrows ) in untreated digits. Nuclei counterstained with DAPI. ( k )
μCT 3-D renderings in Dermabond-treated digits show attenuated bone degradation in response to
Dermabond treatment. Distal is to the left , dorsal is to the top. ( l ) Quantifi cation of bone volume
changes from μCT data of Dermabond treated and control digits, asterisks for signifi cance. ( a – d )
are reprinted from Sammarco et al. [ 7 ], ( e ) is reprinted from Sammarco et al. [ 47 ], and ( f – l ) are
reprinted from Simkin et al. [ 48 ]
5 Digit Regeneration in Mammals