Lingling Sheng

Improvement of the skin flap survival with the bone marrow-derived mononuclear cells transplantation in a rat model.

Partial necrosis of skin flaps remains a significant problem in plastic and reconstructive surgery. In this study we attempted to evaluate the effect of bone marrow‐derived mononuclear cells (BM‐MNCs) transplantation on improvement of skin flap survival in a rat random pattern skin flap model. Thirty Wistar rats were divided into three groups with each consisting of 10 rats. BM‐MNCs and the adipose‐derived stem cells (ADSCs) were transplanted into the subcutaneous tissue in the area where the flap would be dissected. The flaps were then raised two days after cells transplantation. The animals receiving the preoperative Dulbeccos Modified Eagle Medium (DMEM) treatment were used as the controls. On the 7th postoperative day, the survival areas of flaps were measured and tissues were collected for examinations. The results showed that the mean survival areas were 46.33 ± 13.46% in the ADSCs group and 50.06 ± 13.82% in the BM‐MNCs group as the percentages of the total skin flaps, which were significantly higher than that in the control group (26.33 ± 7.14%) (P < 0.05). Histological analysis showed increased neovascularization in the flap treated with BM‐MNCs when compared with ADSCs transplantation. Survival BM‐MNCs and ADSCs were detected in the flap tissues. Higher levels of the basic fibroblast growth factor (bFGF) and vascular endothelium growth factor (VEGF) were found in the BM‐MNCs transplantation group (P < 0.05). The findings from this study demonstrated that preoperative treatment with BM‐MNCs transplantation could promote neovascularization and improve flap survival. These effects of BM‐MNCs on flap survival were comparable with ADSCs transplantation, but without necessity of in vitro cells expansion.

Transplantation of adipose stromal cells promotes neovascularization of random skin flaps.

The delivery of bone marrow-derived mononulear cells (BM-MNCs) has been proved to be effective at promoting neovascularization of ischemic skin flaps. However, the limited source of BM-MNCs restricts their clinical application. Stromal vascular fraction (SVF) contains a group of heterogeneous cells in the adipose tissue, including adipose tissue-derived stem cells, and it has abundant reserve in human body. In this study, we evaluated the therapeutic potential of SVF to promote neovascularization of random skin flaps. Female Wistar rats were randomly devided into three groups with 8 in each group and received allogeneic SVF, BM-MNCs and phosphate-buffered saline (PBS), respectively, before surgery. Two days after cell administration, a 10 × 3 cm random skin flap was elevated. Flap survival, blood flow perfusion and capillary density were examined 7 days after surgery, and the relevant mechanism was also explored. Results showed that SVF group and BM-MNCs group had higher survival percentage (72.2 ± 2.0% and 76.4 ± 3.1%, respectively) as compared with the control group (56.8 ± 4.6%, P < 0.05). Blood flow perfusion and capillary density of flap tissues in SVF and BM-MNCs groups were both improved. The expression levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were increased in flap tissues of SVF and BM-MNCs groups detected by ELISA. These results indicate that SVF could promote vascularization and increase flap survival probably by secreting VEGF and bFGF. The effect of transplantation of SVF on therapeutic angiogenesis of skin flaps is equivalent to that of BM-MNCs.

Stem Cell Therapy for Lower Extremity Diabetic Ulcers: Where Do We Stand?

The impairment of wound healing in diabetic patients is an important clinical problem affecting millions of patients worldwide. Various clinical and basic science studies show that stem cell therapy, as a regenerative medical therapy, can be a good solution. In this paper, we begin with an introduction of the cellular mechanism of the diabetic ulcer. We will then discuss the advantages and limitations of various stem cell therapies that have been under extensive recent study.

Mimic hypoxia improves angiogenesis in ischaemic random flaps

BACKGROUND Hypoxia is the original signal to promote angiogenesis in ischaemic tissues. However, hypoxia-induced angiogenesis usually cannot compensate for the ischaemic injury in surgery, resulting in tissue necrosis. Mimic hypoxia may be an option to improve angiogenesis for the purpose of preventing or reducing necrosis. In this study, the authors explored the feasibility of applying hypoxic mimic--deferoxamine (DFO) to the treatment of ischaemic random skin flaps. METHODS Ischaemic random skin flap models were developed in 18 nude mice using a cutaneous marking technique. The mice were divided into a DFO-treated group and a control group. Vascular endothelial growth factor (VEGF) protein level, vessel density and flap survival rate were evaluated on the seventh postoperative day. In vitro, both VEGF mRNA expression and protein level were investigated in endothelial cells and fibroblasts under DFO, hypoxia and normoxia conditions. The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to study the impact of DFO-induced VEGF up-regulation on endothelial cell viability after severe hypoxia injury. RESULTS The flap survival rate, vessel density and VEGF level in the DFO group are significantly higher than in the control group. In vitro, DFO-induced increase in VEGF mRNA expression translated into 2.3- and 5.8-fold increases in VEGF protein secretion in DFO-conditioned media of endothelial cells and fibroblasts, respectively. The MTT assay showed that the cell viability both in the DFO group and in the VEGF group was significantly higher than in the control group. CONCLUSION VEGF praracrine and autocrine secretion in fibroblasts and in endothelial cells play an important role in DFO-induced angiogenesis, which improves ischaemic flap survival. DFO-induced VEGF autocrine secretion also protects endothelial cells from severe hypoxia injury.

A preliminary study of differentially expressed genes in expanded skin and normal skin: implications for adult skin regeneration

In adults, severely damaged skin heals by scar formation and cannot regenerate to the original skin structure. However, tissue expansion is an exception, as normal skin regenerates under the mechanical stretch resulting from tissue expansion. This technique has been used clinically for defect repair and organ reconstruction for decades. However, the phenomenon of adult skin regeneration during tissue expansion has caused little attention, and the mechanism of skin regeneration during tissue expansion has not been fully understood. In this study, microarray analysis was performed on expanded human skin and normal human skin. Significant difference was observed in 77 genes, which suggest a network of several integrated cascades, including cytokines, extracellular, cytoskeletal, transmembrane molecular systems, ion or ion channels, protein kinases and transcriptional systems, is involved in the skin regeneration during expansion. Among these, the significant expression of some regeneration related genes, such as HOXA5, HOXB2 and AP1, was the first report in tissue expansion. Data in this study suggest a list of candidate genes, which may help to elucidate the fundamental mechanism of skin regeneration during tissue expansion and which may have implications for postnatal skin regeneration and therapeutic interventions in wound healing.

Effect of the PI3K/AKT signaling pathway on hypoxia‑induced proliferation and differentiation of bone marrow‑derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation has been demonstrated to be an effective way of augmenting angiogenesis of ischemic tissue. The low oxygen conditions in ischemic tissue directly affect the biological behavior of engrafted cells. However, to date, the mechanism through which hypoxia regulates self-renewal, differentiation and paracrine function of BM-MSCs remains unclear. Clarification of this mechanism would be beneficial to the use of stem cell-based therapy. The PI3K/AKT pathway has been extensively investigated for its role in cell proliferation, cell transformation, paracrine function and angiogenesis. The present study aimed to analyze the role of PI3K/AKT pathway in hypoxia-induced proliferation of BM-MSCs and their differentiation into endothelial cells in vitro by the application of LY294002, a PI3K/AKT pathway inhibitor, with cells cultured in normoxia serving as a control. The results showed that rat BM-MSCs at passage 3 and 4 displayed only few phenotypical differences in the expression of surface antigens as detected by flow cytometry. When compared with the cells treated in normoxia, the proliferation of BM-MSCs in hypoxia was promoted, a greater number of cells expressed CD31 and a higher expression of vascular endothelial growth factor was observed after culture in hypoxic conditions. However, by inhibiting with LY294002, these changes induced by hypoxia were partly inhibited. In conclusion, the present study showed that the PI3K/AKT pathway served an important role in hypoxia-enhanced in vitro proliferation of BM-MSCs and their differentiation into endothelial cells and paracrine vascular endothelial growth factor.

Cell-Assisted Skin Grafting: Improving Texture and Elasticity of Skin Grafts through Autologous Cell Transplantation.

Background: Full-thickness skin grafts are widely used in plastic and reconstructive surgery. Their poor textural durability and associated contracture make them less desirable than skin flaps. Currently, stromal vascular fraction cells hold great promise because of their angiogenic potential, which may ameliorate the hypoxic period after skin grafting. In this study, autologous transplantation of stromal vascular fraction cells was used in combination with skin grafts to determine whether it improved the texture and other physical property of skin grafts. Methods: Stromal vascular fraction cells were isolated and injected under full-thickness skin grafts in a cohort of 20 rats; a second cohort of 20 rats served as controls. Skin grafts were harvested and analyzed on days 14, 30, and 90 after injections. Bioluminescent imaging with luciferase-stromal vascular fraction cells was used for cell tracing. Contracture ratios, elasticity modulus, and the stiffness of each graft were evaluated. Angiogenesis was evaluated using immunohistochemical techniques against vascular endothelial growth factor. Blood flow signals of the graft were also measured, and expression of vascular endothelial growth factor, hepatocyte growth factor, and basic fibroblast growth factor was assessed in all grafts. Results: Stromal vascular fraction cells markedly decreased the contracture of skin grafts and improved their resilience and elasticity after 1 month. Histologically, the cells enhanced skin thickness and skin vascularization. Moreover, expression of vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor also increased in the stromal vascular fraction group. Conclusion: Autologous stromal vascular fraction cell transplantation enhances angiogenesis after skin grafting and improves the texture and elasticity of skin grafts.

Sodium tanshinone IIA sulfonate prolongs the survival of skin allografts by inhibiting inflammatory cell infiltration and T cell proliferation.

Acute rejection is a major problem for allograft transplantation in the clinic. Classic immunosuppressive drug therapy is accompanied by a variety of side effects. Therefore, safe and effective immunosuppressive drugs remain in demand. In this study, the effect of sodium tanshinone IIA sulfonate (STS) on prolonging the allogeneic skin graft survival was determined using a rat skin transplantation model. Rat recipients were divided into four groups that received different treatments: physiological saline, STS, CsA, or STS+CsA. The results indicated that the administration of STS alone, CsA alone or combined STS and CsA all significantly promoted skin allograft survival as demonstrated by a longer mean survival time (MST) compared with the control group. This effect was due to the reductions in the infiltration of inflammatory cells into allograft and the percentages of CD4+ T cells and CD8+ T cells in the peripheral blood of rat recipients. The injection of STS could also downregulate the expression of RANTES, IP-10 as well as IL-2, IFN-γ and TNF-α in allograft tissue. STS markedly inhibited the proliferation of mouse spleen T lymphocytes stimulated by mitogen and alloantigen in vitro. Taken together, these results suggest that STS is a widely applicable drug with few complications that may serve as a new therapeutic alternative for allograft rejection or even other Th1 cell-dominated immune diseases.

HOXA5 inhibits keratinocytes growth and epidermal formation in organotypic cultures in vitro and in vivo.

BACKGROUND Homeobox transcription factors play important roles in epidermal renewal. Among them HOXA5 emerges as a promising member. However, its direct effect on epidermal biology, either to promote or to inhibit growth, is still controversial. OBJECTIVE We proposed to unravel the role of HOXA5 in modulating keratinocytes growth and epidermal formation in organotypic cultures both in vitro and in vivo. METHODS We transfected HaCaT cells with lentivirual vectors which over-expressed either wild-type or mutant HOXA5 cDNAs with deleted homeodomain. Subsequently we propagated the cells in organotypic cultures (OTCs) and then transplanted them into nude mice. Cell proliferation and cell cycle progression were detected. Epidermal morphogenesis and stratification were investigated by immunohistochemistry and immunofluorescence staining of a series of epidermal markers. RESULTS HaCaT cells transfected with HOXA5 cDNAs displayed lower growth rate and delayed G1-S transition. HOXA5-transfected OTC exhibited an aberrantly organized epithelium with significantly increased TUNEL staining as well as decreased PCNA and K5 staining, while expression of differentiation markers as K10, involucrin and filaggrin were somewhat enhanced. However, under in vivo environment in nude mice which had great paracrine regulatory mechanisms, the aberrant phenotype was ameliorated as shown by a more regular tissue organization and normal expression of PCNA and K5. Inversely, cells transfected with the homeodomain-deleted protein exhibited accelerated growth and produced a more proliferative and better-orchestrated epidermis, as shown by well-expressed proliferation and differentiation markers. CONCLUSIONS HOXA5 can suppress keratinocytes growth and epidermal formation. It probably activated antagonist genes against growth factors release, which depends on its homeodomain.

Foreign body response induced by tissue expander implantation

The foreign body response (FBR) is described as the hosts response to implanted biomaterials, which involves a complex cascade of immune modulators. The dynamic changes of immune cells, inflammatory cytokines and the formation of a fibrous capsule remain to be elucidated. In the present study, a model of subcutaneous implantation of a tissue expander was used. The results revealed that macrophages, the main immune cells in FBR, infiltrated into the expanded tissue and located at the tissue‑material interface from day 1‑90. Following the decrease of the number of macrophages, collagen deposited and fibroblasts transformed into myofibroblasts at the tissue‑material interface, leading to the formation of a fibrous capsule from day 14. The persistent existing macrophages led to a high expression of proinflammatory cytokines, including tumor necrosis factor‑α and interleukin‑1β, both of which initiated the NK-κB and JNK inflammatory pathways, mediating the FBR to tissue expander implantation.