| Cutaneous wounds feature the destruction of the integrity of the normal skin tissue. Because of the lack of the auto-skin, it is soundly difficult for the clinical therapy in efficaciously and quickly reparing the wounds and reconstructing the normal skin tissue for the sake of physiological agglutination of the normal skin function.Mesenchymal stem cells, widly exist in human bone marrow, umbilical cord blood and embryo-precurssor, are originated from mesoderm progenitor and capable of multipotential differentiation, which is thus applied in tissue engineering and cell-replacement therapeutics. The recent study demonstrates that MSCs could also differentiate into epithelium, such as skin, and participate in regenerating and repairing the injuries. UCB is considered as a new alternative sources for MSCs. Compared with that from BM, there are many advantages,such as lower antigenicity, more sufficient source and noninvasive aqcuiring.In our study, MSCs was isolated from the neonate UCB and expanded in vitro. After the cells were labled with 5-bromodeoxyuridine (BrdU), and then adoptively transferred into SCID mice. We addressed the possibility that the UCB-derived MSCs differentiated into epidermic cells and facilitated the wound restoration by detecting the wound consolidation ratio and elucidating the distribution and differentiation status of the donor cells in the newly concrescent skin, which might theoretically and pratically support the clinical application of human umbilical cord blood in wound restoration.Materials and methods:1. Isolation and expansion of MSCs from UCBThe mononuclear cells (MNC) were isolated from the fresh full-term UCB by density gradient centrifugation with Ficoll-Hypaque. The cells were innoculated into the plastic culture flask coated by the FBS and cultured overnight in DMEM/F12 medium supplemented with 15% FBS and 100U/ml Peni-Strep at 37℃,5%CO2 and saturated humidity. The medium was changed completely, to remove the unattached cells. After that, the medium was refreshed every 3 days. Usually after 5-7 days, some cell clones could be found and picked up under microscope. The clones were trypsinizated and cultured in the same condition mentioned above. When the cells grew to 80%-90% confluence, they should be passaged in the ratio of 1:3.2. Identification of the UCB-derived MSCs via flow cytometryThe expanded cells at passage 7 were stained with PE- or FITC-labelled CD29, CD34, CD105 or HLA-DR after, respectively, and the expressions of relative molecules were checked by flow cytometry.3. Proliferative potency of isolated MSCs from UCBThe cultured cell at passage 3 and passage 10 were seeded into the 24-well plates (1×103cell/well) respectively. The medium was changed every 3 days. The cells from two wells were harvested and counted every 2 days till to day 12 after inoculateion. The cell populations were set into longitudinal axis, the timepoints were put into abscissa axis.4. BrdU labelling of UCB-derived MSCs and the detection of the labelling rateThe fresh MSC from UCB at passage 9 were cultured under the same condition with BrdU at the final concentration of 5μmol/L. The medium was changed every 3 days as above. When the cells grew to about 60% confluence, the efficiency of BrdU labeling were assayed by immunocytochemistry. Meanwhile, the proliferateion potencies of the BrdU labeled cells were checked as above.5. Application of cultured MSCs from UCB to repair the full thickness skin defect wound in SCID mice1) Implantion of UCB-derived MSCs directly on the woundSCID mice, male, 4-week-old, were prepared two the full thickness skin defect wounds (1×2cm2) on the both sides of posterolateral thorax. The BrdU labeled MSCs from UCB were collected and washed with PBS, and 5×105 cells in 0.2ml were implanted onto the left wounds of the mice, while PBS was smeared onto the right wound as control. Both of the sides were coverd by vaslin dressing. The size of the remained wounds was measured by asepsis fenestrate membrane on the day 7 and 14 after the operation and the healing rate was caculated by the correlated formula. 2) Injection of UCB-derived MSCs into mice via tail veinAfter the full thickness skin defect wounds on the back of SCID mice was made as above, about 2.5×105 BrdU labeled MSCs from UCB in 0.1ml volume were injected into every mouse via tail vein immediately one by one. PBS was injected into the control group. The wounds were covered with pigskin. There were 8 mice in each group.6. Histological examinationOn the 7th, 14th and 28th day after transplantation, regenerated skin tissue were biopsied from the experimental and control group, The sample were embedded with paraffin and sectioned after fixed with formalin solution. Hematoxylin-eosin (HE) staining and histological observation was according to the standard method.7. Immunohistochemical stainingNewly regenerated skin tissue was biopsied on the 7th, 14th and 28th day after transplantation from the two groups, respectively. The normal skin tissue far away from the wounds, and some internal organs such as heart and liver, were harvested on the 28th day after injecting UCB-derived MSCs via tail vein. All the samples were sectioned as above. The distribution and number of BrdU positive cells were observed and counted after immunohistochemistry (SABC) staining. As far as the section of skin tissue sample, the double positive cells stained with anti-BrdU antibody and anti-Keratin 19 antibody were detected.Results:1. MSCs were isolated from UCB successfully.With the method described above, some clones could be found after the MNCs isolated from UCB were cultured for about 7 to 14 days. The cells picked from the clones could be passaged, and grew like whirlpool as the same morphous as fusiform shape or similar to the fibroblast. It was found by FACS that the cells at 3rd generation could express CD29() and CD105, the positive rates were90.30% and 86.48% respectively, while neither CD34 nor HLA-DR were detectable on the cells.2. BrdU could label the isolated MSC from UCB.It was found the the positive rate of BrdU labeled cells was about 60% by flow cytometry, and the labeled cells grew and expanded as same as non-labeled cells.3. The proliferation characteristics of UCB-derived MSCs in vitro The study found that the eclipse period of UCB-derived MSCs in vitro was 1-2 days after passage. The cells grew into the exponential phase since the 3rd day, and they proliferated actively and got to the peak on the 9th day. After that, the speed of growth slew down and the proliferation entered into the platform stage. There was no significant difference between the MSC proliferative speed and the growth cycle in the various generations, i.e. P3 and P10.4. Application of UCB-derived MSCs to repairing the full thickness defect1). The healing rate was apparently higher in the UCB-derived MSCs implanting group than that in the control(P<0.01), which is (56.06±3.04)% vs (36.99±2.17)% on the 7th day postoperative and (94.75±1.89)% vs (84.63±2.28)% on the 14th day after operation.2). The histological examination elucidated that the wound healing quality in the experimental group looked better than those in the control. In the comparison of the epidermisc layer thickness and the amount of the cells from the newly regenerated skin tissue on the 7th and the 14th day after operation, we found that the epidermisc layer was obviously thicker and the amount of the cells and the dermal ridges was much more in the experimental group than those in the control. Additionally, skin appendages, such as folliculus pili, emerged in the experimental group.3). The distribution of the UCB-derived MSCs in vivo via immunohistochemistryWe were able to detect donor cells derived from umbilical cord blood in the newly regenetated skin tissue at the different time points after operation by UCB-derived MSCs were either implanted on the wound or injected into the mice via tail vein. The BrdU positive cells were found in the basal layer of epidermis, inner layer of folliculus pili, spinous layer and superficial fascia in the newly regenetated skin tissue in the expeimental group. The BrdU positive cells were cube or spherical in the shape and located close to the recipient cells.Furthermore, it was found that there were more the BrdU positive cells located in the basal layer of epidermis in the newly regenerated skin tissue on the 7th day than that on the 14th day after the donor cells injected via tail vein(P<0.01),while there was no significant difference on the 28th day compared with that on the 14th day. Moreover, there were small amount of BrdU positive cells randomly distributing in the normal skin tissue far away from the wound, which was overwhelmingly less than that in the newly regenerated skin tissue. There were also small quantity of UCB-derived MSCs located in the hearts and the livers of the experimental group and mainly existed in the inner-wall of central veins of hepatic lobules and cardiac muscular tissues.5. UCB-derived MSCs differentiation to epidermic (stem) cells in vivoIt was found that there were BrdU positive cells in the basal layer of epidermis and the inner layer of folliculus pili from the newly regenerated skin tissue on the 28th after injecting UCB-derived MSCs via tail vein, and some of them simultaneouslly expressed keratin 19, which meant that some UCB-derived MSCs of the donor might be able to differentiate to epidermic (stem) cells in vivo.Discussion and conclusion1. The protocol that the isolated MNC were innoculated into the culture flask coated by the FBS overnight and the medium was completely refreshed was able to increase the cloning efficiency of MSC, which could eliminate the influence of the osteoclast, facilitate the purification and proliferation of MSC. Finally, MSC derived from the umbilical cord blood of the full-term neonate could be easy and successful obtainment.2. Implantion of UCB-derived MSCs on the wound is able to significantly increase the healing rate and improve the healing quality. The healing wounds in the experimental group looked more similar to the physiological status that the thickness and the cell population of the cuticular layer increase in the newly regenerated skin tissue. Additionlly, it is proved that some donor cells are involved in the formation of newly regenerated appendages of the skinIt can be initially concluded that the UCB-derived MSCs can be induced to epidermic (stem) cells by the microenviroment in the condition of skin injury and the transplantation of UCB-derived MSCs may participate in the repair and reestablishment of the wound by supplementing the repair cell population.3. Very few MSCs were found randomly in the normal skin far away from the wound after injecting via tail vein, while there were a lot of MSC distributing in or around the wound, which indicated that the wound itself could have the chemotaxis to the MSC...
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