Date Published: April 5, 2017
Publisher: Public Library of Science
Author(s): Harshini Sarojini, Adrian T. Billeter, Sarah Eichenberger, Devin Druen, Rebecca Barnett, Sarah A. Gardner, Norman J. Galbraith, Hiram C. Polk, Sufan Chien, Masaya Yamamoto.
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.
Wound healing is a complex and dynamic process involving the replacement of devitalized and missing structures. The traditional view of wound healing is that it involves hemostasis, inflammation, proliferation, and remodeling, and these steps result in a lag of 3–6 d before reepithelialization starts [1,2]. We have discovered that the intracellular delivery of adenosine triphosphate using ATP-vesicles as an acute wound treatment enhances wound healing [3,4]. The most unprecedented finding was that new tissue started to generate within 24 h, and it continued to grow to eliminate the wound cavity quickly [4–6]. This growth was attained by early and massive monocyte/macrophage trafficking, proliferation, and fast collagen production for direct formation of extracellular matrix (ECM). Reepithelialization tunneled through the granulation tissue  and the upper surface of the granulation tissue eventually fell off, revealing a perfectly healed wound. More importantly, the granulation tissue growth had a self-limiting feature, so that no hypertrophic scar formation or any other unusual growth was evident, even after two years [4,6]. This healing process is totally different from the conventional wound healing process, where fibrin, platelets, and red blood cells serve as the main components of the early provisional matrix, which is gradually replaced by granulation tissue during the proliferation phase after a lag of 3–6 d [1,7]. The ATP-vesicle triggered healing process therefore essentially eliminates the traditional lag time and significantly shortens acute wound healing times.
The major findings in this study include the following: 1) intracellular ATP delivery caused rapid tissue regeneration accompanied by early and massive macrophage accumulation that arose as a combination of cell trafficking and in situ proliferation; 2) treatment with ATP-vesicles increased macrophage collagen production whereas the components of ATP-vesicles supplied singly did not show similar effects; 3) macrophage collagen production started very early after ATP-vesicle treatment and continued throughout the study, whereas Regranex treatment increased collagen production only after 48 h of treatment; 4) ATP-vesicle treated macrophages maintained their phenotype and collagen production after 5 d of culture, whereas Regranex treated cells changed their phenotype to fibroblasts after only 48 h of culture; and 5) treatment with ATP-vesicles increased macrophage MCP-1 production but decreased IL-10 production, whereas Regranex treatment produced the opposite effects. The combination of these changes in cell characteristics in response to ATP-vesicles would be expected to result in a very much enhanced healing process in acute wounds.