Random flap is one kind of the most widely used skin flaps in plastic and reconstructive surgery. Its design is limited by the safe ratio of length to width, because of partial distal necrosis of the ultra-long random flap. Many scholars had taken pains to search for feasible strategies for flap survival, such as drugs,1-3 laser,4,5 growth factors,6-8 and cell transplantation.9-12 The success rates in most studies were not so satisfactory that it needs further research.
It had been demonstrated that transplantation of mesenchymal stem cells (MSCs) contributes to improving ischemia through angiogenesis in ischemic vascular diseases.13,14 However, many MSCs died in ischemic sites after transplantation because of the low oxygen concentration in the sites.15,16 Recently, several studies have reported that hypoxic preconditioning could improve survival of MSCs in animal models of ischemic vascular diseases.17,18 Bone marrow mesenchymal stem cells (BMSCs) have demonstrated the potential to differentiate along the bone, cartilage, fat, and muscle lineage. Their harvest from autologous donor sites is relatively harmless and they can readily be cultured in vitro to achieve sufficient cell numbers for transplantation. They have been widely used for tissue engineering and regenerative medicine study.
In the present study, we hypothesized that hypoxic preconditioning BMSCs in a short time can lead to their acquiring the ability to survive for a long time under ischemic condition. As a result, more living BMSCs can promote angiogenesis efficiently and improve the ultra-long random flap survival.
Preparation of cells
Cell acquirement and culture
BMSCs of Sprague Dawley rat (Cyagen Bioscience, USA) were purchased at passage 2. Surface antigens of the third passage cells were examined by Cyagen Bioscience with flow cytometry. The cells were positive for CD44 and CD29 and negative for CD34 and CD45. Osteogenic, chondrogenic and adipogenic differentiation could be induced successfully. BMSCs were routinely cultured and expanded under normoxia (20% O2, 5% CO2) in the whole growth medium, which consisted of low glucose Dulbecco's modified Eagle's medium (DMEM-LG. HyClone, USA) and 10% fetal bovine serum (FBS. GIBCO, Uruguay).
Hypoxic preconditioning of BMSCs
Some BMSCs of the fifth passage were cultured under hypoxia (1% O2, 5% CO2) for 48 hours before transplantation. They were termed hypoxia preconditioned BMSCs (HpBMSCs). The cells cultured under normoxia were termed normoxic BMSCs (nBMSCs).
Cell labeling with carbocyanine 1,1-dioctadecyl-1 conjugated to 3,3,30, 30-tetramethylidocarboyanine perchlorate (CM-DiI) before transplantation
The cells for transplantation were suspended in phosphate buffered saline (PBS, 0.01 mol/L, pH 7.2) at the density of 1×107/ml. Then, they were incubated with CM-DiI at 37°C for 10 minutes and 4°C for 15 minutes. At last they were subjected several times to centrifugation in PBS to remove excess DiI dye and resuspended in DMEM-LG at the density of 8×106/ml. The high labeling rate was observed under fluorescence microscopy and optical microscopy (Figure 1).
Flap model and experimental design
In this study, all animal procedures were approved by the Shandong University Medical College, Institutional Animal Care and Use Committee. All animals were maintained in a specific pathogen-free environment and the surgical procedures were performed under aseptic conditions.
The ultra-long dorsal random flap model was established according to the previous study.12 Thirty-eight-week-old Sprague Dawley rats (average weight (250±30) g) were randomly divided into control group, nBMSCs group, and HpBMSCs group with 10 rats in each. After anesthesia with the sodium pentobarbital, a cranically based ultra-long random flap measured 8 cm ×2 cm was designed on the back of the rat. After incision of the borders, the full thickness flap, consisting of skin and the underling cutaneous maximus muscle and facial layers, were elevated using blunt dissection. In the nBMSCs and HpBMSCs groups, 4×106 labeled cells in 0.5 ml DMEM-LG were injected into the subcutaneous tissue in each rat at 10 points equally following flap elevation. The number was based on the previous study.12 The rats of control group received 0.5 ml DMEM-LG injection by the same way. Similiarly, a 1-mm thick silicone sheet was placed under the flap to prevent angiogenesis from the recipient bed.
After surgery, the rats were raised in their individual cages. No animals died during the study. At day 7 postoperatively, the survival area of each flap was grossly determined based on its appearance, color and texture. The animals were reanesthetized as previously described. Digital images of each flap were recorded with Cannon 1000D (Cannon, Japan), and the area of survival was determined by the ImageJ 1.43 (NIH, USA). Results were expressed as percentage survival in relation to the total surface area of the flap.
After measurement, the flap tissues were biopsied for cell tracking, angiogenesis and enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) level. All the animals were sacrificed by neck dislocation at last.
Ten tissue specimens (1 cm × 1 cm) from each group were collected at the end 1 cm to the demarcation of survival, embedded in optimal cutting temperature compound and sectioned to 6-μm slices for cell tracking under fluorescence microscopy, hematoxylin and eosin (HE) staining and immunohistochemical staining for CD31 (BD, USA) to assess the angiogenesis. A semi-quantitative analysis was performed by measuring microvessel density with CD31 positive reaction on random vision areas of ten slices from each group.
Ten samples (0.5 g) taken from each group were collected from the middle part of the viable flaps, minced in 0.5 ml tissue protein extraction reagent, and then homogenized and centrifuged at 1000 r/min at 4°C for 10 minutes. The supernatant was collected and assessed for VEGF using ELISA kit according to the manufacturer's instructions (R&D, USA). The optical density values of absorbance were read on a microplate reader (BioTek, USA).
The results are expressed as mean ± SD. Comparison between two means was performed by unpaired Student's t-test. Multiple comparisons between three groups were performed by analysis of variance (ANOVA). All data were analyzed using SPSS 14.0 software (SPSS, Inc., IL, USA). Statistically significance was accepted when P <0.05.
The regions of survival and necrosis were clearly demarcated in every flap at 7th day post operation (Figure 2A). The percentages of survival rates were analyzed (Figure 2B). The mean survival rate was (64.4±12.9)% in the HpBMSCs group and (45.7±12.8)% in the nBMSCs group as the percentages of the total skin flaps, which was significantly higher than that in the control group ((24.5±7.1)%) (P <0.05). There was significant difference between the two cell groups (P < 0.05).
Cell survivalin vivo
It was confirmed by cell tracking in vivo under fluorescence microscopy that more labeled BMSCs survived in the ischemic flaps of the HpBMSCs group, when compared with the nBMSCs group (Figure 3). HE staining of the tissue sections showed that more cells scattered in cutaneous maximus muscle and fascial layers where they were injected in the HpBMSCs group than in the nBMSCs group. There were the fewest cells scattered in the control group (Figure 4).
Neovascularisation in flaps of both the nBMSCs and HpBMSCs groups was improved. It was evidenced by the exaggerated amounts of microvessels marked by CD31 in sections, which were taken from surviving regions close to the necrosis-survival margin. Microvessel density (MVD, microvessel number per high power field, ×400) was significantly higher in both the HpBMSCs and nBMSCs groups (36.20±8.19 and 30.01±5.68) when compared with the control group (17.60±4.19) (P <0.05). The MVD in the HpBMSCs group was also significantly higher than that in the nBMSCs group (P <0.05) (Figure 5).
VEGF level in flaps
The VEGF protein level was found significantly higher in the tissues from the HpBMSCs group ((300.05±50.41) pg/g) than those from the nBMSCs group ((240.55±33.64) pg/g) and the control group ((191.65±32.58) pg/g) (P <0.05). Difference was also significant between the nBMSCs group and the control group (Figure 6).
Random skin flap is a simple method for skin deficiency and widely used. It can be used without any specific problem as long as the ratio of length to width is around 1.5:1 to 2:1. However, if the ratio is higher, complications such as partial necrosis may occur. The neovascularization in the skin layer is the most important factor for the survival of an ultra-long random flap. It is a complex process, which involves the proliferation of vascular endothelia cells and the cooperation among various growth factors.
Apart from many drugs,1-3 physical methods4,5 and growth factors,6-8 a few kinds of cells have been used to improve flap survival, such as endothelial progenitor cells,9 bone marrow-derived mononuclear cells12 and MSCs.10-11 MSCs are multipotent cells and can serve as primary scaffolds and secrete protective humoral factors. BMSC was widely studied for tissue engineering and regenerative medicine. MSCs have been shown to be highly immunosuppressive.19,20 Although the exact mechanism is uncertain, many scholars had applied allograft or xenograft of MSCs for studies,12,21 and so we did.
One major problem of MSCs therapy was that a large number of cells died after transplanted into the ischemic sites,15,16 which may reduce the effect of therapy. As evidenced by the low oxygen level of specimens aspirated from bone marrow volunteers, BMSCs live in relative hypoxic physical conditions in vivo.22 Hypoxia can improve MSCs' survivability, proliferation and maintain their stemness.14 Some investigators have applied hypoxic preconditioning of embryonic stem cells and MSCs to study,18,23 however, the effect of HpBMSCs on ultra-long random flap survival has not been reported.
The present study displayed that HpBMSCs took better effect on improvement of ultra-long random flap survival than nBMSCs. Cell tracking of the transplanted BMSCs labeled with CM-DiI in vivo showed that more HpBMSCs survived in the ultra-long random flap than nBMSCs. Immunohistochemical staining for CD31 antigen reflected the angiogenesis in the flaps of each group. Microvessel density was the largest in HpBMSCs group and the least in control group. The VEGF protein levels displayed the same change trend. The more BMSCs survived in vivo, the more microvessels and the higher VEGF level were detected. From above we can deduce that the final result of improving ultra-long random flap survival attributes to the higher survival rate of HpBMSCs in the ischemic flap. Some studies had got the similar result that more HpBMSCs can survive in ischemic condition to take biological effects.18,23
CD31 antigen is an immunologic marker for vascular endothelia cells. The immunohistochemical staining result showed that more vascular endothelia cells were located in the flap. Were they differentiated from BMSCs or recruited from peripheral blood? We can not explained this because the restriction of our work. However, some scholars had found the evidence that MSCs can differentiate into vascular endothelia cells to contribute to angiogenesis.10,24 This may back our deducement that more HpBMSCs survived in ischemic flap and take biological effects for angiogenesis.
Paracrine of growth factors is an important biological effect of MSCs. It had been confirmed that they can promote angiogenesis indirectly through the release of growth factors.24 Although VEGF is the strongest for angiogenesis, one deficiency of our study was that we had not examined the other growth factors which must take effects, such as fibroblast growth factor-2 and epidermal growth factor. The other limitation was that we preconditioned BMSCs under 1% O2 for 48 hours before transplantation, which may be not the most perfect preconditioning density or time. It needs further study.
In summary, HpBMSCs transplantation improves ultra-long random skin flap survival in rats. The mechanism of therapy may be explained by that more HpBMSCs could survive in ischemic condition to take biological effect for angiogenesis. This provides a novel and promising strategy for the application of BMSCs transplantation in ischemic flap, because hypoxic preconditioning is a simple and safe procedure.
The authors thank Professors LIU Shao-hua, HU Ying-wei, SUN Shan-zhen, QU Xun for their guidance in the design of this study.
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Keywords:© 2011 Chinese Medical Association
random skin flap; cell transplantation; mesenchymal stem cells; hypoxic preconditioning; angiogenesis