Figure 1.
Skin temperature differences between the non-ischemic ears and ischemic ears.
The difference was larger at the beginning of the experiment, but decreased gradually over time. However, a temperature difference of more than 1°C was maintained at the end of one month.
Figure 2.
Comparison of complete reepithelialization times between wounds treated with ATP-vesicles and those treated with Regranex.
Figure 3.
Comparison of wound cavity size reduction rate in wounds treated with ATP-vesicles and those treated with Regranex.
(A) Non-ischemic wounds; (B) Ischemic wounds.
Figure 4.
A representative comparison between the wounds treated with ATP-vesicles and Regranex. Granulation growth starts to occur one day after surgery in non-ischemic wounds when treated with ATP-vesicles. In ischemic wounds, the granulation occurs a few days later, but wounds treated with ATP-vesicles still heal faster than those treated with Regranex.
Figure 5.
More comparisons of granulation tissue.
All ATP-vesicle treated wounds start to generate granulation tissues within 1–3 days while the wounds treated with Regranex show no such growth.
Figure 6.
Representative photographs and H&E staining wounds at day 3 of treatment.
The wound treated with ATP-vesicles produces new tissue growth that has completely covered the wound bed and overtaken the wound edge (arrow). The growth is filled with cells. The wound treated with Regranex produces a hypertrophic rim of tissue, which sometimes grows inward (40x, arrows).
Figure 7.
A photomontage of wounds and microscopic changes occurring within 24 hours after surgery.
When ATP-vesicles are used, granulation starts to appear within 12 hours (A), and H&E staining indicates a rich cellular component (B). Granulation tissue growth continues and covers the whole wound at 24 hours (C). Granulation tissue shows positive anti-MAC387 staining (D), which is further confirmed by CD163 staining (inset) (E). PCNA staining indicates very active proliferation of these cells (F), which is further confirmed by BrdU antibody staining (inset). Wounds treated with Regranex do not display this rapid growth.
Figure 8.
Comparison of macrophage numbers in the early days of the healing process.
Regranex-treated wounds show a slight increase in macrophage numbers. However, when ATP-vesicles are used, macrophage numbers are 7–8 times higher than in the Regranex-treated wounds at day 1, but decrease quickly thereafter.
Figure 9.
Macrophage accumulation in early days.
When the wounds are treated with ATP-vesicles, solid granulation occurs at day 3 (B). The growth is filled with macrophages, which occur not only in wound cavity, but also underneath the ear cartilage (D). The wounds treated with Regranex do not have similar growth (A, C).
Figure 10.
Comparison of Regranex and ATP-vesicle treated wounds 3 days postoperatively.
Rich collections of macrophages are stained dark brown to black by Anti-Mac387 (top panels), and more collagen is shown by van Gieson stain (low panels) in the wounds treated with ATP-vesicles. Regranex treated wounds do not show a similar effect.
Figure 11.
Comparison of direct collagen fiber counts 3 days after surgery.
Wounds treated with ATP-vesicles show 4–5 times higher collagen content than those treated with Regranex.
Figure 12.
Comparison of cell survival in culture.
The addition of ATP-vesicles (1 mM Mg-ATP) into the culture medium increases monocyte-macrophage survival time 3–4 folds compared to the culture medium alone, Regranex (0.001%), or LPS (100 ng).
Figure 13.
Dose-dependent collagen production by macrophages.
Macrophage collagen production appears to be dose-dependent when ATP-vesicles are used. The higher the ATP concentration, the more collagen 1α1 is produced.
Figure 14.
Most of the macrophages still maintain their original phenotype, whereas culturing with Regranex causes fibrocyte transformation. Inset shows the photos of the culture treated with ATP-vesicles (A) and treated with Regranex (B).
Figure 15.
Appearance of healed wounds treated with ATP-vesicles.
A representative sequential photomontage showing the smoothness of the healed wounds treated with ATP-vesicles even two years after treatment.