AFAIK, we currently don't understand how chakra regeneration works. Oro said we filter chakra from the environmental mixture:
"So when a ninja uses chakra…" Hazō said.
"They are solely using a single phase of chakra. A human's chakra coils filter human chakra from the ambient mixture – analogous to lifting a stone up to the top of a hill. It creates tension and latent movement. The boulder wishes to roll down the hill and gains speed and power by doing so. Its movement can knock down trees if released correctly. In the same way, filtered chakra within a human's coils can be returned to the ambient mixture in such a way as to produce useful effects. This naturally disproves the explanation provided to you of nature chakra – were it simply a broader set of components from the ambient mixture, it would require less work to separate, and would thus release less energy when used in ninjutsu. Instead, nature chakra releases more energy when used."
However, Oro may not actually be right about this description. Chakra doesn't seem to obey conservation of energy in the way this would suggest. Take Elemental Mastery - the EM nuke works because the chakra effect instantly cools any gas entering the AOE, regardless of how much gas that is or how much work it has to do. That's not the behaviour of something with a limited amount of energy to expend before it runs out. MEW is another point against Oro's explanation; it's possible to create matter
ex nihilo with less than 40 CP. If Oro's hypothesis were correct and there was a limited amount of energy associated with chakra returning to the ambient medium, that limit would have to be
vast, and chakra coils would have to do a correspondingly vast amount of work. (Oro himself obviously doesn't believe this; but Oro doesn't know about the EM nuke, and doesn't know that matter should require vast amounts of energy to create. And it may not, in MfD world; but together with the EM nuke working it's highly suggestive.)
So I think Oro's hypothesis, at least the part about phases returning to the ambient mixture, is probably
not correct. Since we don't know what experiments he did to get to the things he told us and can't judge their validity, we should also consider the possibility that his model for chakra regeneration is flawed.
Hence this rune. It's a very simple test; turn on the rune, have someone spend their chakra and sit around in the AoE, and see if their chakra regenerates. It's something Oro may not have been able to do; this quote is weak evidence for that:
"Ambient chakra is a mixture of numerous subtypes of chakra, which, apart from human chakra for humans and nature chakra for summons, are currently impossible to interact with, survey, and study. All humans possess an affinity for human chakra, though only ninja are capable of using it effectively."
He may well just have meant that the individual subtypes are impossible to interact with, but a literal reading of this statement says that he can't manipulate ambient chakra, in which case he may not have been able to test whether or not it's required for chakra regeneration.