Fig 1.
Comparison of wound reepithelialization between ATP-vesicles and Regranex.
When 50% of wound was reepithelialized in the non-ischemic wounds, it took an average of 8 days for ATP-vesicles while 10.5 days for Regranex (p = 0.00026, 1A). In the ischemic wounds, the average 50% reepithelialization time for ATP-vesicles was 11.2 days vs. 16.1 days for Regranex (p = 0.0001, 1B). Because reepithelialization often tunnels through the granulation tissue in the wounds treated by ATP-vesicles (1C), the actual reepithelialization in these wounds might be faster.
Fig 2.
A representative comparison of wound granulation and healing between the two groups.
In the wounds treated by ATP-vesicles, new tissue starts to appear only 24 hours after surgery in the non-ischemic wounds. In the ischemic wounds, generation of granulation tissue is slower. However, the wound still heals faster than that treated by ATP-vesicles.
Fig 3.
Wound cell comparison at day 1 after treatment.
(A): The wound treated with ATP-vesicles has already been covered by newly generated tissues. Anti-PCNA and BrdU stains show very actively proliferating cells in ATP-vesicle treated wounds when compared to Regranex treated wounds. The brown color indicates DAB-positive immunostaining (scale bar = 50 μm). (B): Bar graph showing the differences between the two groups.
Fig 4.
Comparison of the picrosirius red staining at days 4 and 15.
(A): The wound treated with ATP-vesicles shows a higher volume of collagen (bright red), while the control wounds have little collagen. The picrosirius red staining is viewed under circular polarized light. Increased type I (orange) and type III collagen (green) content is seen in ATP-vesicle-treated wounds as healing progresses. The control wounds have much less collagen expression. The collagen fibers also increase in size and mature at a faster rate in ATP-vesicle-treated wounds when compared with the controls (Scale bar is 100 μm). (B). Graphical representation of morphometrically measured area represented by collagen type 1 among the three groups.
Fig 5.
(A) FACS analysis to determine the purity of monocytes isolated from human blood. The isolated cells were CD-14 and HLA-DR positive. The purity of the isolated cells was 98%. (B): Time response effect of various treatments on collagen production in human macrophages. *p<0.05 ATP-vesicles vs. Regranex and medium. The monocyte/macrophage (500,000 cells/ml) cells were treated with ATP-vesicles (1 mM Mg-ATP) and controls for 3, 6, 12, 24, and 48 h. The collagen type 1α1 levels increased with time when the macrophages were treated with 1mM ATP-vesicles when compared with macrophages treated with Regranex or culture medium alone.
Fig 6.
(A): Anti-mac387 and collagen type 1α1 immunostaining on monocyte/macrophage cells treated with 1 mM ATP-vesicle or Regranex for 5 d. Cells treated with medium alone didn’t survive for 5 d. Green color represents anti-mac or type 1α1 collagen staining and blue is the DAPI staining for the nucleus. Collagen type 1α1 immunostaining is higher in ATP-vesicle treated cells when compared with the controls (scale bar = 50 μm). (B). Graphical representation of the cells having macrophage phenotype following treatments.
Fig 7.
Effect of ATP-vesicles and Regranex on MCP-1, IL-10 and IL-6 production in cultured human monocyte/macrophages.
(A-C): The release of MCP-1, IL-10 and IL-6 in culture supernatant was measured by ELISA method. Data are the mean ± SD of six independent experiments (*p<0.05).
Fig 8.
Sircol collagen assay on monocyte/macrophage cells treated with different doses of ATP-vesicles.
Soluble collagen production measured by Sircol assay (mean ± SD of six independent experiments, *p<0.05). (A): Human cells. (B): RAW264.7 mouse macrophage cells.
Fig 9.
Hematoxylin-eosin staining shows the macrophage morphology in the cells treated with ATP-vesicles.
Both the macrophage cells maintain macrophage morphology when treated with ATP-vesicles. (A): Human cells. (B): RAW264.7 mouse macrophage cells.
Fig 10.
A schematic illustration of possible mechanisms that could explain the rapid tissue regeneration seen in response to intracellular ATP delivery: 1) treatment of ATP-vesicles causes massive macrophage accumulation in the wound wall and cavity; 2) the treatment also cause macrophage in situ proliferation; 3) the supplied energy supports cell survival and function in the wound cavity even though neovascularization has not completed; 4) the activated macrophages produce collagen directly; 5) neovascularization is enhanced, which directly transforms the early cell mass into the extracellular matrix; and 6) reepithelialization is enhanced on the basis of rapidly formed granulation tissues. In the control wounds, cell migration and accumulation cannot be achieved because there is no energy supply in the wound cavity.