Rapid tissue regeneration induced by intracellular ATP delivery—A preliminary mechanistic study

Abstract

We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery—extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.

Uptake of polyphosphate microparticles in vitro (SaOS-2 and HUVEC cells) followed by an increase of the intracellular ATP pool size

“…endothelial cells during tube formation in vitro [ 15 ] or also during wound healing in experimental animal studies in vivo [ 17 ], is the result of the uptake and subsequent metabolic processing of polyP microparticles. The present study of polyP microparticles and the observed growth of the intracellular ATP pool also impressively confirms a recent study, where ATP (encapsulated into nano-/microparticles) initiated and maintained rapid tissue regeneration [ 18 ].…”

“…These data strongly suggested that not only an intracellular ATP rise but also an exposure to polyP result in an intensified (neo)vascularization. It is a constructive coincidence that almost simultaneously with our report on the acceleration of the wound healing process by application of polyP microparticles [ 17 ] an independent report was published that indicated that such a rapid wound regeneration can also be initiated and completed by encapsulated ATP [ 18 ]. As main causative factors for this rapid tissue regeneration, both macrophage activation and neo-vascularization had been assumed [ 18 ].…”

“…It is a constructive coincidence that almost simultaneously with our report on the acceleration of the wound healing process by application of polyP microparticles [ 17 ] an independent report was published that indicated that such a rapid wound regeneration can also be initiated and completed by encapsulated ATP [ 18 ]. As main causative factors for this rapid tissue regeneration, both macrophage activation and neo-vascularization had been assumed [ 18 ]. In light of the data give here it can be comprehensively and unifyingly been proposed that intracellular ATP, either provided via the intermediary metabolic network and/or the mitochondrial oxidation steps coupled with ATP formation or–as proposed here–through metabolic energy transfer from polyP to ATP promote wound healing.…”

“…Likewise we could demonstrate in vivo that polyP, if fabricated as nanoparticles [ 16 ], causes in mice a marked acceleration of wound healing, in particular in diabetic experimental animals [ 17 ]. Simultaneously and in parallel with our approaches it has been published that ATP, if delivered intracellularly after encapsulation into vesicles, induces a very rapid process of tissue regeneration in an experimental animal system (rabbits), which started already after 24 h [ 18 ]. This effect has even been demonstrated in diabetic animals [ 19 ] and is of clinical importance since it is well established that the energy supply in diabetic tissue is reduced and wound healing is impaired [ 20 ].…”

Modulation of adenine phosphoribosyltransferase-mediated salvage to promote diabetic wound healing

“…These concentrations of energy metabolites in the wounds of patients with diabetes were significantly lower than those of normal patients (Smith, Mills et al, 1999). Given that energy is required for every aspect of the wound healing process, many strategies sustain the cellular ATP level by encapsulated ATP or blocked ATP loss would be developed as medications for morphogenetically active wound-healing formulations (Muller, Wang et al, 2017, Sarojini, Billeter et al, 2017.…”