Innovations_in_Molecular_Mechanisms_and_Tissue_Engineering_(Stem_Cell_Biology_and_Regenerative_Medicine)

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In general, tissue vascularity plays a critical role in maintaining physiological

function and homeostasis. Although there are a few tissues that are inherently avas-

cular, e.g. cornea and articular cartilage , most of the body depends on the vascula-

ture for distributing nutrients and blood bound signals as well as removing waste

products. In this context, the avascular digit blastema exists as a structure that is

largely isolated from the physiological infl uences of the body. One consequence of

this is that the blastema creates a physically less turbulent microenvironment that

might be more conducive for effective long range cell-cell signaling involving

secreted factors (e.g. BMPs , WNTs, FGFs, etc.), some of which are known to play

essential roles in the regenerative response. In this context, enhanced revasculariza-

tion might physically disrupt intercellular signaling between blastema cells and thus

contribute to the failed regenerative response. The observation that blastema forma-

tion occurs following VEGF or BMP9 treatment is consistent with this hypothesis.

On the other hand, the avascular microenvironment also limits the availability of

essential nutrients, such as oxygen, to blastema cells and this would create a hypoxic

microenvironment. Indeed, recent studies document that the blastema is hypoxic,

and that oxygen availability during the regenerative response is dynamic [ 7 ].

Oxygen tensions change dynamically in temporally and spatially distinct and

predictable patterns during P3 regeneration. In histological samples, hypoxic

regions are identifi ed by immunohistochemical localization of injected pimonida-

zole ( Hypoxyprobe-1 Plus ) that forms stable adducts in regions of less than 1.3 %

oxygen, and hyperoxic regions are identifi ed immunohistochemically based on the

presence of FBLX5 , a protein that is stabilized at oxygen levels greater than 5.5 %

[ 7 ]. During digit regeneration, hyperoxic conditions remain relatively constant and

are predominantly associated with the vasculature, consistent with the conclusion

that vasculature plays a role in limiting oxygen availability. The development of a

very prominent, but transient, hypoxic zone is observed during stages of blastema

formation, and that zone dissipates with the initiation of re-differentiation

(Fig. 5.6a–d ). To test the requirement of the hypoxic blastema microenvironment on

the regeneration process, mice were exposed to Hyperbaric Oxygen (HBO) treat-

ment , targeting the period of blastema formation. A single HBO treatment is suffi -

cient to disrupt the hypoxic microenvironment of the blastema, but regeneration is

not inhibited by either targeted or continuous HBO treatment [ 7 , 47 ], thus the

hypoxic microenvironment of the blastema is not required for successful regenera-

tion. However, there is clear indication that HBO treatment does induce specifi c

modifi cations of the regeneration process. HBO treatment enhances the activity of

osteoclasts during the histolytic phase resulting in an extended period of bone deg-

radation and a delay in blastema formation (Fig. 5.6e ) [ 47 ]. This suggests that while

a hypoxic blastema is not a requirement for regeneration, cells involved in regen-

eration are responsive to changing oxygen tension and this plays a role in regulating

phase transitions during the regenerative process. The interaction between osteo-

clasts and osteoblasts has been studied in the context of bone turnover and bone

diseases, such as osteoporosis and osteopetrosis , and regulatory pathways have

been identifi ed. Osteoclasts are derived from monocytes and express Receptor

Activator of Nuclear Factor kβ (RANK) , while osteoclastogenesis during

L.A. Dawson et al.