Autologous and not allogeneic adipose-derived stem cells improve acute burn wound healing

<div><p>Adipose-derived stem cells (ADSCs) transplant has been reported to be a potential treatment for burn wounds. However, the effects of autogenicity and allogenicity of ADSCs on burn wound healing have not been investigated and the method for using ADSCs still needs to be established. This study compared the healing effects of autologous and allogenic ADSCs and determined an optimal method of using ADSCs to treat acute burn wounds. Experiments were performed in 20 male Wistar rats (weight, 176–250 g; age, 6–7 weeks). Two identical full-thickness burn wounds (radius, 4 mm) were created in each rat. ADSCs harvested from inguinal area and characterized by their high multipotency were injected into burn wounds in the original donor rats (autologous ADSCs group) or in other rats (allogenic ADSCs group). The injection site was either the wound center or the four corners 0.5 cm from the wound edge. The reduction of burn surface areas in the two experimental groups and in control group were evaluated with Image J software for 15 days post-wounding to determine the wound healing rates. Wound healing was significantly faster in the autologous ADSCs group compared to both the allogenic ADSCs group (<i>p<0</i>.<i>05</i>) and control group (<i>p<0</i>.<i>05</i>). Wound healing in the allogenic ADSC group did not significantly differ from that in control group. Notably, ADSC injections 0.5cm from the wound edge showed significantly improved healing compared to ADSCs injections in the wound center (<i>p<0</i>.<i>05</i>). This study demonstrated the therapeutic efficacy of ADSCs in treating acute burn wounds in rats. However, only autologous ADSCs improved healing in acute burn wounds; allogenic ADSCs did not. This study further determined a superior location of using ADSCs injections to treat burn wounds including the injection site. Future studies will replicate the experiment in a larger and long-term scale burn wounds in higher mammalian models to facilitate ADSCs therapy in burn wound clinical practice.</p></div

Comparison of burn wound healing rates between different ADSC injection sites.

<p>(A) Burn wound healing rates on different days in the group treated by injection of autologous ADSCs at the wound center (Auto Center), the group treated by injection of autologous ADSCs in the four corners 0.5cm from the edge of the wound (Auto 0.5cm), and the group treated by injection of K-NAC medium at the wound center (Control). Data are shown as mean ± S.E.M. (Number of animals = 3; Number of samples = 6). *Significant difference of Auto 0.5cm from Control (<i>p</i><0.05). ^#Significant difference of Auto 0.5cm from Auto Center (<i>p</i><0.05). <i>p</i> values were calculated by <i>paired t- test</i>. (B) Burn wound healing rates on different days in the group treated by injection of allogenic ADSCs at the wound center (Allo Center), the group treated by injection of allogenic ADSCs in the four corners 0.5cm from the edge of the wound (Allo 0.5cm), and the group treated by injection of K-NAC medium at the wound center (Control.) Data are shown as mean ±S.E.M. (Number of animals = 3; Number of samples = 6).</p

Wound healing rate of no treatment VS control.

<p>Wound healing rate of no treatment VS control.</p

Full-thickness contact burn wound.

<p>The H&E staining results for burn wounds created by varying durations of contact (30, 40, 50, and 60 seconds) with a heated metal rod. In the 30 second group, the full-thickness of the skin was burned but the muscle layer was intact. White arrow indicates the panniculus carnosus. Note the damaged muscle layers (inflammation in 40 and 50 seconds groups; necrosis in 60 seconds group) in the wounds created by contact longer than 30 seconds.</p

Localization of ADSCs in wound by Qtracker^® labeled- ADSCs.

<p>(A-C) The H&E staining results 7 days after injection of autologous ADSCs at the four corners, 0.5cm from the wound edge. (A) 40X magnification, (B) 100X magnification, and (C) visualization by Qtracker^® labeling under 100X magnification. (D-F) The H&E staining results 5 days after injection of allogenic ADSCs at the four corners, 0.5cm from the wound edge. (D) 40X magnification, (E) 100X magnification, and (F) visualization by Qtracker^® labeling under 100X magnification. The Qtracker^® labeled- ADSCs are indicated by blue arrows.</p

Comparison of burn wound healing rates between autologous and allogenic ADSCs treatment.

<p>Burn wound healing rates on different days were calculated for the group treated by injection of autologous ADSCs at the wound center (Auto Center), the group treated by injection of allogenic ADSCs at the wound center (Allo Center), and the group treated by injection of K-NAC medium at the wound center (Control.) Data are shown as mean ± S.E.M. (Number of animals = 3; Number of samples = 6). *Significant difference from Control (<i>p</i><0.05). ^#Significant difference from Allo Center (<i>p</i><0.05). <i>p</i> values were calculated by <i>paired t- test</i>.</p

Experimental design.

<p>Experimental design.</p

Fat graft regulated p-AKT/AKT and Bax/Bcl-2 ratios in the spinal cord dorsal horn.

<p>(<b>A</b> and <b>B</b>) Both expression ratios of p-AKT/AKT and Bax/Bcl-2 increased significantly in Group C versus Group D. These results indicated that fat grafting reduced cell death of neuron in the spinal cord (Data were presented with mean ± SEM, **<i>P</i> < 0.01; *<i>P</i> < 0.05).</p

Fat grafts increased p-IκB, reduced the p-NFκB protein, and through the inhibition of p-JNK, attenuated apoptosis in the spinal cord dorsal horn.

<p>(<b>A</b>) Double immunofluorescent staining was performed to measure p-IκB and p-NFκB. Few p-IκB proteins were expressed in Group C, but p-IκB were increased in Group D. The expression of p-NFκB was abolished and reduced by fat grafts in Group D versus Group C. (<b>B</b>) Double immunofluorescence images in p-JNK and TUNEL expression were elevated following an increase in the TUNEL-positive cells. In Group D, we found that the inhibition from fat graft of p-JNK attenuated apoptosis in the spinal cord dorsal horn.</p

Fat grafting inhibited the expressions of inflammatory proteins, COX-2, iNOS and nNOS, in the spinal cord dorsal horn, and reduced the secreted levels of pro-inflammatory cytokines, IL-1β and TNF-α.

<p>(<b>A</b>) Western blot analyses of COX-2, iNOS and nNOS in the spinal cord dorsal horn at 8 weeks after the burn injury. β<b>-</b>actin was used as the internal control. The presentation format is similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137563#pone.0137563.g002" target="_blank">Fig 2</a>. Protein expressions of COX-2, iNOS and nNOS were decreased significantly in the burn with fat graft group compared with the burn with saline injection group (Group D vs. Group C). (<b>B</b>) Fat grafts appreciably reduced the secretions of the pro-inflammatory cytokines, IL-1β and TNF-α, in the spinal cord dorsal horn. IL-1β and TNF-α assessment was conducted at least in triplicate, n > 3, *<i>P</i> < 0.05.</p