Cell Therapy Improves Flap Prefabrication By Transplanting Vascular Endothelial Growth Factor Modified Adipose-derived Stem Cells

Part I Cell therapy improves flap prefabrication by transplanting adipose-derived stem cellsBackground:The reconstruction of head and neck after serious injuries is a challenge in plastic surgery, owing to its high demands of aesthetic outcomes and limited option of donor sites. Skin graft can hardly achieve satisfied results due to skin contraction and abnormal colors, while free skin flaps are usually limited by its blood supply and tissue types. Flap prefabrication starts with transposition of a vascular pedicle into a body of donor tissue that otherwise does not possess an axial blood supply. Ideally we can build an axial pattern skin flap by combining any axial blood vessels with any random pattern skin flaps through flap prefabrication, which could possibly allow any defined tissue volume to be transferred to any specified recipient sites, and then fit any complex reconstruction demands in head and neck injuries.Although the application of the prefabricated flap has become a promising approach to repair complex defects, the risk of partial necrosis limits its potential for clinical applications due to the unpredictable neovascularization manually produced by transferring a vascular pedicle. Formation of a prefabricated flap depends on the process of neovascularization and tissue regeneration between the vascular carrier and the donor tissue. In order to promote this process, several means have been explored to achieve better neovascularization. Such as gene therapy and the administration of angiogenic cytokines.Cell therapy is a technology that uses autologous, allogeneic or xenogeneic somatic living cells, whose biological characteristics are altered by manipulation or not, to transplant to patients for therapeutic, diagnostic or preventive purpose. Recently, following the development of stem cell research, cell therapy has been extended to the areas of biological or immunological treatments toward tumors, diabetes, or nervous system diseases. It can also be used to assist the revascularization of transplanted tissue and infarcted cardiac muscles, or to promote wound healing. By now, bone-marrow stem cells, adipose-derived stem cells and endothelial progenitor cells all have been proven feasible to promote revascularization of the ischemic tissue in animal models. The most intensively studied adult stem cell is the bone marrow derived mesenchymal stem cells, but the limited source of bone marrow restrained its applications. On the other hand, adipose-derived stem cells (ASCs) can be harvested abundantly from lipo-aspirates of liposuction surgeries in plastic surgery clinics and they have been considered ideal in many aspects for cell therapy:they can be harvested, handled, and multiplied noninvasively, easily, and effectively; their pluripotency and proliferative efficiency are revealed to be the same as bone marrow-derived stem cells. Therefore, ASCs are especially suitable for applications of cell therapy in plastic surgeries. Here we hypothesized that topically injected ASCs could promot flap prefabrication and tested this hypothesis in a rat prefabricated flap model.Obiective:To investigate the feasibility of using adipose derived stem cells as a source of cell therapy to improve flap prefabrication.Methods:1. Isolation and characterization of rat ASCs.Adipose tissues were harvested from the inguinal fat pads of inbred male Wistar rats. They were excised, finely minced, and digested with 0.1% collagenase for 60 minutes at 37℃with vigorous shaking. Cell suspension was centrifuged and the cell pellet was resuspended with Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Then, the isolated cells were seeded onto culture plate and incubated at 37℃in 5% carbon dioxide. The first medium change was at 24 hours after seeding and then the medium were replaced every 3 days in the following culture. Cells were harvested at 80 to 90 percent confluence and passaged at a ratio of 1:3. At passage 2, the cultured cells were digested and resuspended in PBS at a concentration of 2×106cells in 0.5 ml for injection into each prefabricated flap.Subconfluent ASCs of passage 2 were cultured with specific induction media to demonstrate their multilineage differentiation capacities. After cultured in adipogenic, osteogenic and chondrogenic media for four weeks respectively, multilineage differentiation of ASCs were confirmed using histological and immunohistological assays.2. Isolation of rat chondrocytes.The articular cartilage was harvested from inbred male Wistar rats and then cut into 2x2mm2 slices. After being digested with 0.25% trypsin plus 0.02% EDTA at 37℃for 30 min, the cartilage slices were further digested with 0.1% collagenase in DMEM at 37℃for 12-16 h. Then, chondrocytes were harvested, counted, and seeded onto culture dishes in DMEM with 10% FBS. Cells were harvested at 80 to 90 percent confluence and passaged at a ratio of 1:3. Chondrocytes of passage 2 were used for the following experiments.3. Rat prefabricated flap model. We performed two-stage operation on inbred male Wistar rats to produce an abdominal prefabricated skin flap:At stage one, an abdominal rectangle peninsular flap, based caudally, was elevated from the xiphoid process cranially and the pubic region caudally to the anterior axillary lines bilaterally. The right femoral artery and vein were dissected with the preservation of a 0.3-cm wide muscle around the vessels to avoid vessel injury and to keep collateral arteriovenous communication. The femoral vessel bundle was ligated distally at the level of the ankle joint and then transposed through a subcutaneous tunnel. It was implanted underneath the abdominal rectangle flap along the diagonal line. The bilateral superficial epigastric artery and vein were ligated to induce ischemia in the abdominal flap. After that, the implantation site around the pedicle was injected with rASCs (2×106cells in 0.5 ml PBS) as group A (n=12), with 0.5 ml PBS as blank control group B (n=12), and with rat chondrocytes (2×106cells in 0.5 ml PBS) as negative control group C (n=12). The flaps were then sutured back to their native configuration.Four weeks after the first-stage operation, the abdominal island flap based on the implanted vascular pedicle was elevated along previous incisions. Then, the pre-fabricated flap was sutured in place without dressing.4. Assessment of flap survivalThe total area of the prefabricated island flaps were measured immediately after the second-stage surgery. While on postoperative day 7 when flap necrosis were readily identified, survival area of the flaps were assessed in a blinded fashion by two independent specialists with respect to the gross appearance, color, and consistency of the flaps and the presence or absence of cutting bleeding. The total and survival areas of the flap were portrayed on semitransparent papers, and then analysed by MOTIC image system (Motic Images Advanced 3.2, Motic China Group CO., LTD.). Results were expressed as percentage survival. 5. Neovascularization assessment by capillary densityTissue sections from the same position (around the distal end of the embedded vascular pedicle) of the surviving part of the flap were harvested, embedded in paraffin for conventional hematoxylin and eosin histological assessment and von Willebrand Factor (vWF) immunohistochemical staining. Neovascularization was assessed by measuring the numbers of vWF-positive capillaries in 10 randomly chosen fields of each slide (100×magnification).The counting was performed by two blinded reviewers.6. Detection of angiogenic cytokine VEGF in vitro and in vivo.At passage 2 after cell seeding, the supernatant of ASCs cultures were collected at various time points up to 10 days for enzyme-linked immunoassay (ELISA) of rat VEGF.We performed the first-stage operation on another 54 rats, which were also randomly divided into ASC group, chondrocyte negative control group, and PBS blank control group. After surgery, we harvested the prefabricated flap on day 1, day 4 and day 7 postoperative. Six flaps of each group were harvested at each time points. Then subdermal soft tissues from four sites of each flap were harvested and processed with ELISA assay to detect local concentrations of VEGF.7. Statistical analysis.All results were expressed as mean±SD (Standard Deviation). One-way analysis of variance (ANOVA) followed by least significant difference (LSD) test was used to compare flap survival percentage and capillary density data from the three experimental groups. Repeated measures ANOVA analyses (with Bonferroni confidence interval adjustment) were used and conducted for data of the VEGF ELISA assay. A p-Value<0.05 was considered statistically significant. All statistical analyses were performed using SPSS(?) version 16.0 software. Results:1. Cultured rat ASCs displayed a fibroblast-like morphology, with strong proliferative ability in vitro. They can be passaged for the first time 7 days after seeding, and then passaged every 3 to 4 days subsequently. After cultured in lineage-specific medium for 4 weeks, adipogenic, osteogenic and chondrogenic differentiation of rat ASCs were confirmed by Oil Red-O staining, alizarin red staining and collagenⅡimmunohistochemical staining respectively, which confirmed multilineage differentiation capacity of the cultured rat ASCs.2,After the first operation, no flap necrosis or contraction was observed. However, after the second operation, necrosis inevitably occurred at the distal region of the prefabricated flap. During the second surgery on the subcutaneous side of the flap, a vasoganglion around the distal end of the embedded vessel pedicle could be observed under laser speckle view, which was supplied by the implanted vessel. A significant increase in flap survival was observed in rats receiving ASCs, as compared to the control groups:30.71±6.99 percent in ASC group versus either 19.90±4.40 percent in PBS group or 17.53±4.38 percent in chondrocyte group (p<0.001). Meanwhile, there's no statistical difference of the survival percentage of the flap between chondrocytes group and PBS group (P>0.05).3. Quantitative analyses revealed that capillary density of ASC group was significantly higher than that of the control groups (27.85±13.64 capillaries per mm2 in ASC group vs.10.18±5.74 capillaries per mm2 in PBS group and 7.63±4.24 capillaries per mm2 in chondrocyte group, P<0.001). Furthermore, there was no significant difference in capillary density between chondrocyte group and PBS group (P>0.05).4. ELISA assay of VEGF protein demonstrated that the supernatant fluid of in vitro cultured ASCs contained VEGF, which was definitely secreted from ASCs. Furthermore, VEGF level in the supernatant revealed time dependent increase within 7 days after passage and then underwent a little decrease after that.In vivo detection of VEGF levels indicated that at the 4th and the 7th day after the first operation, ASCs treated group had a statistically significant higher level of VEGF (1665.77±323.49 and 2821.82±654.88pg/ml respectively) compared with the PBS group (923.20±115.54 and 1190.00±400.33pg/ml respectively, p<0.001) and chondrocyte group (193.40±32.57 and 266.45±103.50pg/ml respectively, p<0.001). And VEGF levels in chondrocyte group was significantly lower than that of the PBS group at day 4 (193.40±32.57 vs 923.20±15.54 pg/ml, p<0.001) and day 7 (266.45±103.50 vs 1190.00±400.33pg/ml, p<0.05). Furthermore, in vivo VEGF level in the prefabricated flaps also displayed time-dependent increase in ASC group after the first operation. However, the same tendency couldn't be observed in the control groups, and the chondrocyte negative control group even occured slightly decrease.Part II Vascular Endothelial Growth Factor gene transfection enhances therapeutic effect of ASCs promoting flap prefabrication.Background:The application of angiogenic cytokines to protect ischemic tissue and to promote revascularization have been reported for a long time, but they all have a very short action time and are easy to run off. Gene therapy targeting some angiogenic agents such as VEGF was also proven to be helpful for improving angiogenesis, however the severe side effects of viral vectors restricted its clinical applications. So neither the local application of angiogenic cytokines nor the administration of gene therapy can be long-term effective without obvious side effects.On the other hand, cell-mediated gene transfer, which can also be considered as gene modified cell therapy, may display a better results in improving neovascularization than single use of either cell therapy or angiogenic cytokines.Thus we transduced ASCs with VEGF to see if its angiogenic effect could be augmented through gene modification.Vascular Endothelial Growth Factor (VEGF) is the strongest inflammatory and angiogenic agent, which could induce angiogenesis and endothelial cell proliferation and stimulate vascular permeability. It has been shown to enhance revascularization of ischemic tissues, including skin flaps, when applied topically and injected intravenously. The different isoforms of VEGF have been purified on the basis of their affinity to heparin. In human, VEGF165 (a 165-amino acid isoform) is the most physiologically active one. Thus we transduced rat ASCs with human VEGF165 gene to see if combining ASC cell therapy with VEGF gene therapy could achieved a better result than using ASCs or VEGF alone.Objective:To testify if the angiogenic effect of ASCs on flap prefabrication could be augmented through VEGF gene modification.Methods:1. Construction of recombinant adenoviral vectors.Adenovirus carrying VEGF 165 (Ad.VEGF165) and adenovirus carrying green fluorescence protein (Ad.EGFP) were constructed by AdEasy adenovirus system. This part of work was majorly carried out by Institute of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, P.R. China.2. Adenoviral infection.To select the best multiplicity of infection (MOI) for adenovirus-mediated gene transfer, ASCs were exposed to Ad.EGFP at MOI 10,20,50, and 100 for 24 h. Flow cytometry was used to quantify the transfection efficiency of the adopted MOI according to the expression of green fluorescent protein (GFP). The optimal MOI in the following experiments was chosen for both the highest GFP expression and cell viability.3. Human VEGF 165 protein expression after gene transfection. ASCs were transfected with Ad.VEGF165 at the optimal MOI.The secretion of VEGF165 by transfected and untransfected rat ASCs was determined by ELISA assays.ASCs transfected with Ad.EGFP were processed with MTT assay compared to the untransfected ASCs to determine whether adenoviral transfection could damage cell proliferation activities.4. Rat prefabricated flap model.The surgical procedures were the same as partⅠ, in this group, each rats were injected with 2×106 ASCs transfected by Ad.VEGF165. Flap survival and capillary density were compared with data in part I.Results:1. The cytopathic effect was obvious in ASCs transfected with Ad.EGFP at MOI 100, while the adenoviral transfection did not influence the growth characteristics of ASCs at MOI 10,20 and 50. On the other hand, the FCM assay displayed that when transfected at MOI values higher than 50, the transfection efficiency didn't increased tremendously. Thus we chose 50 as our optimal MOI for the following experiments.2. After transfected with Ad.EGFP, the expression of green fluorescent protein in rat ASCs could be observed under fluorescence microscopy. The MTT assay of transfected and untransfected ASCs showed no obvious difference, which indicated that the adenoviral vector couldn't damage cell proliferative activities under optimal MOI.After transduced by Ad.VEGF165, we detected human VEGF165 protein in cell culture supernatant of rat ASCs, The VEGF165 level increased after gene transfection and peaking on day 6.3. Rat prefabricated flap model of the Ad.VEGF165 transfected ASC group showed higher ratio of flap survival compared with the ASC group. The capillary density counting also revealed the same result. These results means the VEGF gene modification can enhance the therapeutic effect of ASCs for promoting flap prefabrication.summary:1. Our experiments transplanted ASCs to a rat prefabricated flap model to enhance flap prefabrication and observed positive results:the survival percentage of the prefabricated flap increased when treated with ASCs, the capillary density also increased at the same time. On the other hand, flaps treated with chondrocytes didn't reveal similar results compared with the blank control group. As a result, we can draw a conclusion that locally injected ASCs could increase flap prefabrication.2. We detected the most typical angiogenic cytokine VEGF in vivo and in vitro and found that in vitro cultured ASCs could secrete VEGF. The in vivo VEGF level among experimental groups also displayed statistical differences:those in ASC group were significantly higher than those in the control groups, and the VEGF level in the chondrocyte negative control group were statistically higher than in the PBS blank control group. These results indicated that the therapeutic effects of ASCs may correlate to their angiogenic cytokine secretion such as VEGF.3. According to these findings, we transduced rat ASCs with human VEGF165 gene by adenoviral vector and administrated this transfected ASCs into our prefabricated flap model. Eventually, we found the therapeutic effect of the transplanted ASCs enhanced as the survival percentage and capillary density of the flap both increased when compared to ASCs alone.