Random flaps are one of the most commonly used methods to repair soft tissue defects caused by trauma or tumor ablation (Eto et al., 2013). However, ischemia and necrosis are the most serious complications associated with the repair process. Thus, the main purpose of current studies is to develop an effective method to promote the survival of ischemic tissue and achieve clinical translation.
ADSCs are considered an ideal type of stem cell for promoting tissue regeneration and repair. However, ADSCs lose the surface marker CD34 after subculture, which limits the efficacy of this treatment. Stromal vascular fractions (SVFs) are highly heterogeneous populations of cell hybrids obtained after digestion of adipose tissue and contain both CD34 + ADSCs and other cell subpopulations expressing CD34. Moreover, SVFs did not need subculture, which can avoid the influence of the subculture process on the expression of CD34 markers. Thus, in our study, we attempted to use CD34 + cells isolated from SVFs to promote the survival of ischemic tissue and hypothesized that the effect was superior to that of ADSCs.
We used random axial flaps in nude mice to evaluate the efficacy of adipose-derived CD34 + cells and ADSCs in protecting tissue from necrosis. Both CD34 + cells and ADSCs showed significant effects in promoting tissue survival compared to CD34- cells. When compared to each other, the survival rate in the CD34 + Group was significantly higher than that in the ADSC Group. Histological staining showed more collagen deposition and less inflammatory cell infiltration in the CD34 + group than in the ADSC group, indicating that the tissue structure was better preserved in the CD34 + group.
As the ex vivo results showed, the protective effect of adipose-derived CD34 + cells may involve two mechanisms: promoting vascularization and ameliorating inflammation.
Vascularization is vital to ischemic tissue (Ju, Wu, & Hou, 2016). Histological analysis of ischemic skin showed that tissues from the CD34 + Group contained more CD31 + cells than those from the ADSC Group. ADSCs have been reported to have angiogenic effects by secreting VEGF, bFGF and other growth factors (Ferrara & Alitalo, 1999; Selvaprithviraj, Sankar, Sivashanmugam, Srinivasan, & Jayakumar, 2017). In our ex vivo study, we found that adipose-derived CD34 + cells expressed higher levels of VEGF and bFGF mRNA than ADSCs, as well as the related secreted proteins. The in vivo results showed that higher VEGF and bFGF expression at both the mRNA and protein levels was observed in skin tissue harvested from the CD34 + group. Moreover, we found that SDF-1 was significantly more highly expressed in CD34 + cells than in cultured ADSCs. SDF-1 is an important chemokine that can induce cell migration and promote angiogenesis (Bromage et al., 2019; Lee et al., 2013). The expression of SDF-1 was also significantly higher in tissues from the CD34 + group than in those from the ADSC group. SDF-1 can promote EPC migration and proliferation via the PI3K/AKT signaling pathway. In HUVECs, the CM of CD34 + cells promoted more capillary tube formation than that of ADSCs, indicating that adipose-derived CD34 + cells had a better angiogenic effect than ADSCs.
Reduced immune cell infiltration was observed in skin sections from the CD34 + group. Fewer CD68 + macrophages were found in the CD34 + group than in the other groups. Ischemia induces the secretion of inflammatory factors, leading to inflammatory activation and worsening tissue necrosis. Previous studies have reported that ADSCs can ameliorate inflammation (Maumus et al., 2011). In our study, CD34 + cells secreted higher levels of the anti-inflammatory factors IL-10 and TGF-β and lower levels of the proinflammatory factors TNF-α and IL-1b than ADSCs. We also found that the expression of p38 MAPK and NF-κB was reduced in tissues from the CD34 + group. The p38 MAPK/NF-κB pathway initiates a cascade reaction via phosphorylation, resulting in the release of proinflammatory cytokines (Ju et al., 2016; Wang et al., 2017). CD34 + M2 macrophages, which play a role in regulating inflammation (Pan et al., 2015), may contribute to this effect. Therefore, adipose-derived CD34 + cells had a better effect on ameliorating ischemia-induced inflammation than the other cells.
Compared with ADSCs, CD34 + cells have the advantages of rapid isolation and no need for long-term in vitro culture, showing promising prospects for clinical transformation. Our research demonstrated that adipose-derived CD34 + cells show better efficacy in promoting the survival of ischemic tissue than ADSCs. However, this study also has some limitations. This study assessed the effect of CD34 + cells on the survival of random axial flaps using nude mice. Clinical trials are needed to evaluate the clinical efficacy of adipose-derived CD34 + cells. Moreover, to determine whether the cells persist when transplanted after injection, we need to perform follow-up studies.