Abstract
BackgroundPartial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. The purpose of this study was to determine whether Tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability.MethodsPrimary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for 2 weeks. Cell Counting Kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Polymerase chain reaction and western blot analysis were used to assess the expression of β-catenin, vascular endothelial growth factor (VEGF), sex determining region Y-box 2 (SOX2), OCT4 (also known as POU domain class 5 transcription factor 1), Nanog, and glycogen synthase kinase-3 beta (GSK-3β) in TSA-treated cells. In other experiments, after mice were pretreated with TSA for 2 weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was conducted to examine Wnt signaling as well as stem cell- and angiogenesis-related biomarkers in epithelial tissue in vivo.ResultsEpidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2, Nanog, and OCT4 were also higher in TSA-pretreated epithelial cells than in control cells. In the reproducible ischemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and VEGF and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue.ConclusionsTSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.
Highlights
Partial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue
Epithelial skin cells showed enhanced resistance to hypoxia after Tanshinone IIA (TSA) pretreatment DAPI staining showed that isolated epithelial skin cells that were pretreated with TSA (2 mg/L) for 2 weeks exhibited enhanced resistance to Cobalt chloride (CoCl2)
The viability of control epithelial cells declined after 72 h of treatment with CoCl2, whereas epithelial cells pretreated with TSA showed higher viability (Figure 2A)
Summary
Partial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. Flaps are routinely used in plastic surgery for covering tissue defects resulting from trauma, ablative surgery, or congenital malformation, partial or total flap necrosis after flap surgery is not uncommon [1,2]. More research is needed into stem cell transplantation and gene therapy before experimental results can be translated into broad clinical application [9,10]. As flap transplantation is becoming more widely used in reconstructive surgery, it is important to understand the mechanism of flap necrosis so that it can be prevented. When hypoxia exceeds the tolerance level of epithelial cells, necrosis or scarring of flap tissue occurs. Stem cells are defined by their capacity for self-renewal and their resistance to maladaptive microenvironments, including hypoxia, and there is compelling evidence that multipotent stem cells contribute to recovery from hypoxia and epidermal repair [13,14]
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