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

EPO attenuates burn-induced motor neruon apoptosis.

<p>(A) Expression levels of BCL-2, BAX, and cleaved caspase-3 in the spinal cord ventral horn, measured through Western blotting. (B) Significant reduction in BCL-2/BAX expression was observed in Group B compared with Group A (100%). However, BCL-2/BAX expression was upregulated after EPO treatment. (C) Cleaved caspase-3 expression increased in the burn-induced group compared with the sham-control group. Similar to the notable reduction in cleaved caspase-3 expression following EPO treatment, a decrease in cleaved caspase-3 was observed. *: p < 0.05.</p

EPO alleviated burn-induced muscle cells apoptosis by Western blotting.

<p>Burn injury significantly increased the expression of cleaved caspase-3 in muscle cells vs Group A (p < 0.01), and Groups C and D exhibited decreased cleaved caspase-3 expression (p < 0.05) vs Group B.</p

Immunofluorescence staining results for detecting activated microglia expression in the spinal cord ventral horn.

<p>(A) Increased expression of activated microglia was observed in Group B compared with Group A. EPO administration resulted in lower microglia expression in Groups C and D compared with Group B (scale bars: 100 μm). (B) Quantitative analysis of activated microglia revealed a significant increase in the number of activated microglia in Group B compared with Group A. EPO treatment attenuated microglia activation. Values are expressed as a percentage of the mean ± SEM (n = 6). *: p < 0.05; **: p < 0.01.</p

Grouping and flow chart of the animal study procedure.

<p>Sham burn or burn injury was induced at day 0. Following the procedure, wound care with 1% SSD was repeated twice a day. EPO was given for pharmacological investigation in Group C and D with different regimens. After completion of the protocol, all rats were sacrificed on week 1 (W1). Spinal cord ventral horn and gastrocnemius muscles were taken and processed by histopathological examination or Western blotting. Abbreviation: D, day; W, week.</p

Effect of EPO on hematological parameters.

<p>Effect of EPO on hematological parameters.</p

Wound healing rate of no treatment VS control.

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

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

Immunofluorescence staining images and quantitative analysis of COX-2 and iNOS in the spinal cord ventral horn.

<p>The immunofluorescence staining of COX-2 (A) and iNOS (B) 7 days after burn injury. The quantitative analysis of COX-2 (C) and iNOS (D) from spinal cord tissues in each experimental group has been assessed. COX-2 and iNOS expression decreased in the EPO-treated groups (Groups C and D) compared with the untreated group (Group B). *: p < 0.05; **: p < 0.01.</p

The Effects of EPO on autophagy markers in burn injury model by immunofluorescence analysis.

<p>(A, B) Double immunofluorescence staining and merged images using LC3B, ATG5, and NeuN in the spinal cord ventral horn were shown. The quantitative analysis of ATG5 (C) and LC3B activity (D) were measured. Burn injury significantly increased LC3B and ATG5 immunoreactivity in Group B versus Group A. After EPO treatment, LC3B and ATG5 immunoreactivity decreased markedly in Groups C and D vs Group B (**: p < 0.01; *: p < 0.05 versus Group B; Group A: 100%). Scale bars: 50 μm.</p