| Objective:To investigate the feasibility of transplantation of mesenchymal stem cells into expanion skin.Methods:The femurs of the porket were harvested in asterile fashion and the marrow was flushed with phosphate buffered saline (PBS).The mononuclear cell layer of the density medium's upper layer was collected to begin the culture. Passage 3 BM-MSCs were resuspended in PBS. Cell aliquots were incubated with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies specific for CD29, CD90, CD34 and CD45.Cells cultured were analyzed by fluorescence-activated cell sorting.Sixteen pigs divided into four groups randomly with four each. In Group A. each flap received suspended stem cells around the wound. In Group B, stem cells in medium were injected into the ear vein. In Group C, each wound received medium and Group D was the blank control group.After hair removal from the dorsal surface and anesthesia, six flaps were marked on either side of the spine midline by injection of ink in dermal and the area was 4cm×4cm.The 80 ml silicone expanders were implanted under the flap as described previously. The animals were injected the normal saline (N. S.) (10 ml every three days until day 28).The marked area was measured at day 7,14,28 after the expansion. After the expanders were removed, the area was measured immediately. Skin tissue specimens taken after 28 days of expansion were tested the content of vascular endothelial cells, proliferating cell nuclear antigen, and the epidermal stem keratinocytes. They were identified with an antibody against CD31,PCNA and K19 respectively, compare the positive cells between four groups.Skin tissue specimens taken after 14 days of expansion were tested the target gene expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor-1 (SDF-1) by real-time PCR.Result:Mesenchymal stem cells appeared morphologically to be ahomogenous population of fibroblast shaped cells. The results showed that the BM-MSCs did not express CD34 nor CD45. However, almost all the cells expressed CD29 and CD90.With the expansion prolonged, the marked area in each group increased. Compared to Group D, the marked areas of Groups A, B and C increased significantly (P<0.01). Measurements of the marked area after removing the expander showed that Groups A, B, and C all had different degrees of retraction, and the remained areas of Group A and Group B after retraction were still larger than that of Group C. Group A's was statistically larger than Group C's (P <0.05).BM-MSCs labeled with CM-DiI were observed throughout the subcutaneous fascial layer of flaps on day 7,14, and 28 after the implantation. There was obviously distribution in group A. With the extending of expansion, the cells labeled with CM-DiI increased in group B, which were mainly distributed in the dermis. None CM-DiI-Labeled BM-MSCs were detected in groups C and D. The vascular cross section densities (article/field) of groups A, B, C and D were 9.1±2.31,7.85±1.93,6.5±1.64,4.2±1.54 respectively. PCNA staining showed that positive cells in group A were 5-6 layers under the base of Epidermis, while there were 2-3 layers for group B and there were only scattered single cells for group C. K19 staining showed that under the base of epidermis there were full thickness positive cells for group A and part of the stratified region, while the continuous cells distribution were seen in group B, and there were only scattered cells in group C. Real-time quantitative PCR test results showed the multiples of difference in target gene expression of VEGF, SDF, EGF and bFGF were 5.4,1.07,4.03 and 6.03 times respectively for group A, as compared with group B.Conclusion:1,Transplant of BM-MSCs into local or systemic of expansion model can promote the growth of biological skin.2,local transplantation achieves better results than systemic transplantation. |
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