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.

Sarojini H, Billeter AT, Eichenberger S, Druen D, Barnett R, Gardner SA, Galbraith NJ, Polk HC Jr, Chien S. Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study. PLoS One. 2017 Apr 5;12(4):e0174899. doi: 10.1371/journal.pone.0174899. PMID: 28380006; PMCID: PMC5381896.

ABSTRACT

BACKGROUND: Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery and were tested for diabetic skin wounds in rabbits.

METHODS: Diabetes was induced by alloxan. The mean peak blood glucose concentration was 505 mg/dL. One ear was made ischemic and 80 full-thickness wounds were created in 10 animals. ATP-vesicles or saline was used and healing was compared.

RESULTS: On the non-ischemic ears, mean closure time for ATP-vesicles-treated wounds was 13.7 days versus 16.4 days for saline-treated wounds (P < .05). On the ischemic ears, mean closure time for ATP-vesicles-treated wounds was 15.3 days versus 19.3 days for saline-treated wounds (P < .01). Histological study indicated better healing and re-epithelialization in the ATP-vesicles-treated wounds.

CONCLUSIONS: Intracellular delivery of ATP accelerated the healing process of diabetic skin wounds on ischemic and non-ischemic rabbit ears. The mechanisms deserve further study but may be related to improved cellular energy supplies.

Wang J, Wan R, Mo Y, Li M, Zhang Q, Chien S. Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits. Am J Surg. 2010 Jun;199(6):823-32. doi: 10.1016/j.amjsurg.2009.05.040. PMID: 20609726; PMCID: PMC2901881.