In the present study, a rat model with a mean full-thickness burn area of 38 ± 4 % of TBSA was generated by immersing the rat dorsum in 100°C water for 15 s. Without therapeutic intervention, the model exhibited a 40 % mortality rate, simulating a clinical picture of severe burn injuries. Given that data regarding the relationship between burn size and mortality in small mammals is lacking [28, 29], it is always challenging to create a regular and uniform reproducible burn model for mortality study. The variations in the burn depth in different model designs and post-burn care protocol resulted in different mortality rates as reported in the literature. While one study demonstrated 62.5 % mortality rate in rats with burns covering 26–30 % of TBSA [30], the others reported 10 % mortality in a 30–40 % of TBSA burn model [31, 32].
Large burns cause an elevation of serum cytokines levels which have been demonstrated both in humans and animals after thermal burns and associated with mortality. Our results showing that 77.8 % (7/9) of mortalities occurred in the first week after the onset of burn injury was compatible with the statement that the serum IL-6 levels peaked during the first hours after burn injury and were proportionate to the burned size [33]. In animal studies, the serum levels of proinflammatory cytokines were found to increase from days 2 to 7 after infliction of burns of varying degrees [8, 34]. The elevated serum cytokines cause an increase of systemic capillary permeability resulting in protein leakage into the interstitial space, generalized edema, and eventually hypovolemic shock [35, 36].
The present study proved that intravenous infusion of hcMSC attenuated SIRS in large burns. Although some might argue that there was a lack of evidence in serum cytokine level measurement, our study results were supported by cytokine data from other similar studies. Carolina et al. (2016) demonstrated that intradermal subcutaneous injections of MSCs altered plasma cytokine levels in burned rats [37]. Using a 30 % TBSA burned rat model, Liu L. et al transplanted 5×106 GFP-labeled human umbilical cord mesenchymal stem cells (HUCMSCs) at day 3 after burn via a tail vein injection [22] and concluded that HUCMSCs remarkably decreased the quantity of infiltrated inflammatory cells and levels of IL-1, IL-6, TNF-α and increased levels of IL-10 and TSG-6 in wound. In another study, a reduction in the plasma levels of proinflammatory cytokines IL-6, IL-1β and TNF-α was proved to result in a low mortality rate [21].
Although the immune modulation and immune suppression properties of MSCs have been proved in animal studies, their bio-distribution following intravenous injection is a critical issue of discussion. As they are relatively large cells and express various adhesion molecules, our previous study has shown that the majority are trapped within capillaries of various organs, especially in the lungs before the cells reach their target following injections. Although Liu L. et. al mentioned that HUCMSCs migrated to the burn wounds two weeks after injection via the tail vein, Su, LJ et al., by injecting albumin-conjugated fluorescent nanodiamonds (FNDs) pcMSCs via internal jugular vein in a miniature pig for quantitative tracking, mentioned that 80 % of the injected pcMSCs was found in the lung 24 h after intravenous delivery, and decreased to 75 % after a week [38]. Based on a previous study result showing that elevated IL-1 beta was found in the lung tissue after severe burns [39], the entrapment of hcMSCs in lung has become a therapeutic advantage in treating the deteriorating pulmonary functions caused by cytokine storms [40]. Because of the bioactivities of hcMSCs in the wound [37], we assumed that the entrapped MSCs in the lung decrease neutrophil and macrophage infiltration, as well as proinflammatory cytokine production including levels of IL-1, IL-6 and TNF-α [38, 39, 41, 42], confirming the beneficial effects of the intravenous infusion of hcMSCs on the severely burned rats.
The present study proved that a single dosage of 2 × 106 hcMSCs intravenous infusion was sufficient to have beneficial effects on the burn outcome. Due to the absence of standardized dosage, the dosage used in the present study was based on our previous animal study [40]. As over-dosage may lead to pulmonary embolism and the problem of progressive cell apoptosis may affect efficacy, further investigations are necessary for the study of optimal dosage based on burn severity, half-life of the infused hcMSCs, and body weight of the recipient. Moreover, some may question our choice of using hsMSCs in the study. The reason was because hsMSCs was the only source of stem cells available in our stem cell culture research laboratory. Its advantages include ready availability as a waste product of delivery, low major histocompatibility complex molecule expression, ease of cell isolation and culture, and no requirement of invasive surgical procedures in the donors. However, the associated risk of protumorigenic effects is the main complication that deters its future clinical applications [22, 43], although some argue that the risk is relatively low in hsMSC compared to adipose or bone marrow derived MSCs. Further investigations are necessary to assess the safety and efficacy of the hsMSC treatment and our study results will be useful for the design of future translational researches on infusion stem cell therapy in extensive burns.
The major limitation of the present study was the lack of data showing the impact of hcMSC treatment on serum cytokine levels, although previous studies in the literature have proved that hcMSC decreased the serum cytokine levels. Another drawback was the decrease in sample size resulting from successive mortalities. In addition, in the study, late mortality after 14 days was not investigated and the status of wound healing was not assessed.