| IntroductionIn the treatment of bone injury, bone coloboma and bone ununion have been the key problems. Traditional operation possesses great injury and limited bone and a better operation is needed. In rescent studies, tissue engineering technology has been the role treatment of bone coloboma and bone ununion, and transplantation therapy using mesenchymal stem cells (MSCs) has been the research focus. In papers, the focus was bone marrow-derived mesenchymal stem cells (BMSCs), but not human umbilical cord mesenchymal stem cells (hUC-MSCs). The main researches about hUC-MSCs are the culture, identify, induced differentiation and gene modification, minor about the mechanism of hUC-MSCs in transplantation therapy of bone injury."Homing" is the general characteristic of MSCs as well as hUC-MSCs. After injury, exogenous MSCs homing toward injured part and inflammatory tissue, but the lower survival rate and relevance rate of MSCs hamper the treatment effect. And further reseatch is needed for the mechanism of MSCs homing. From papers, we found that tissue injury, transplanted pathway, transplanted time, transplanted number and culture in vitro influent the homing of MSCs. Transplanted pathway contains venous transplantation (relative safe), arterious transplantation (indued possibility of micro artery occlusion), endocardial transplantation and local transplantation (direct injection into target organ).Which pathway is the most effective and safe? There is no answer. And further study is needed in venous transplantation and local transplantation. Furthermore, amplification in vitro of MSCs could reduce the C-X-C chemoking receptor (CXCR) and reduced CXCR diminishes the homing of MSCs. In a number of animal tests, gene modification could promote the living, homing and differentiation of MSCs and improve its effect, but gene modification elevates the risk of ectopic bone formation and tumor formation. And virus used in this process is lack of safty. Wether transplantation after differentiation could improve the homing and living of hUC-MSCs needs further study.Now it has been proved in tests in vitro that hUC-MSCs could secrete momocyte chemotactic protein-1(MCP-1), interleukin-6(IL-6), tissue matrix metalloproteinases inhibitor-1, interleukin-8(IL-8). The cytokines above play an important role in tissue injury and repair. So we have a hypothesis:wether hUC-NSCs could secrete same cytokines in the injury part; wether bone induction in vitro could promote paracrine function of hUC-MSCs. In order to confirm our hypothesis, we did some works.In researches, we found that bone morphogenetie proteins (BMPs) as the most important cytokines in bone injury and repair played critical role in the differentiated stage of osteoprogenitor cells to osteoblast (OB) as well as various stages of bone repair. When bone injury happened, BMPs binds to specific receptor on the membrane and activates multiple proteins related to bone formation through smads pathway. The concentration of BMPs is positively correlated to the level of bone repair. In this research, we study that wether different transplantation and differentiation of hUC-MSCs could promote bone repair.Number of homing hUC-MSCs and microenvironment regulation are the main factors which affects the ecffet of hUC-MSCs transplantation. In this research, Wister mice were used as the model of bone injury. Homing of hUC-MSCs and the expression of BMP-2were detected after different transplantation and osteogenic induction in order to find the effective method which could improve homing of hUC-MSCs and bone repair. Objective1. To detect the influence on homing and survive of hUC-MSCs by venous transplantation, local transplantation and osteogenic induction in vitro.2. To detect the expression of BMP-2in bone injury part by venous transplantation, local transplantation or osteogenic induction in vitro of hUC-MSCs.Methods1. Separation, culture and detection of hUC-MSCsObtain hUC-MSCs from umbilical cord of healthy fetus through tissue adherent method. The cells in culture in DMEM media with10%fetal bovine serum and digested by trypsin-EDTA. And detect the expression of CD73,CD105,CD34and CD45of the third generated cells.2. Osteogenesis induction and detection of hUC-MSCsInduced media (0.1μM dexamethasone,50μM ascorbic acid,10mM beta-glycerophosphate sodium,10%FBS and DMEM media) of osteogenic differenti ation is added into the sixth, fifth or third hUC-MSCs. Change media every three days, and transplantation is done at the third, seventh or fourteenth day.Alizarin red stain:three weeks after osteogenic differentiation, paraformaldehyde is added, followed alizarin red, and the cells were observed under a microscope.3. Bone injury model of Wistar miceAdult Wistar mice were anesthetized through intraperitoneal injection, unilaterally or bilaterally expose distal femur, and horizontally cut the metaphysic deep to marrow cavity.4. Tests of homing hUC-MSCs in vivoImmunohistochemical stain for hUC-MSCs using mouse anti-human nucleimonoclonal antibody (MAB1281) after venous injection or local injection in the section of bone injury, the stem cell are observed under a microscope.5. Dectetion of BMP-2in bone injury of Wistar miceBMP-2is the key cytokine in the process of bone repair. In the research, Immunohistochemical stain for BMP-2(BA0585) in the section of bone injury, and the positive cells were observed under a microscope.6. Experimental groupsIn study for homing of hUC-MSCs, Wistar mice divided randomly into venous injection groups and local injection groups,further divided by no differentiation, osteogenic induction for3days,7days or14days. Injection of saline is as a control. In study for BMP-2, wistar mice divided into3groups:venous injection groups; local injection groups; control groups. The first two groups contain4subgroups:no differentiation, osteogenic induction for3days,7days or14days.7. Systemic inflammatory response in bone injury model ratsDivided into hUC-MSCs transplantation treating bone damage model group, nothing treating bone damage model group and healthy rats control group.At the7th and14th day after hUC-MSCs transplantation, venous blood is abtained by tail vein after abdominal anesthesia.Rats in the nothing treating bone damage model group are drawn blood at the same time. Healthy rats are directly drawn blood.All blood specimens are send to the Fouth People’s Hospital,to check routine blood test,to compare white blood cells and red blood cells in all groups.Results1. Immunohistochemical stain for hUC-MSCs at the7th day after cell transplantation, plantation of hUC-MSCs in local injection group was more than in venous injection group. There was no difference among no differentiation group,3days group,7days group and14days group, and no plantation of hUC-MSCs in control groups.2. Immunohistochemical stain for hUC-MSCs at the14th day after cell transplantation, hUC-MSCs survived more in3days group and7days group than that in no differentiation group and14days goup. Local injection group induced significantly survival of hUC-MSCs compared with venous injection group.3. At the7th and14th day after cells tranplatation,white blood cells are not different between hUC-MSCs transplantation treating bone damage model group and nothing treating bone damage model group, different with control group. Red blood cells are not different in all groups. 4. Immunohistochemical stain for BMP-2at the7th day after cell transplantation:no significant difference among no differentiation group,3days group,7days group and14days group; BMP-2positive cells were more in local injection group than that in venous injection group; BMP-2positive cells were more in experimental group than that in control group.5. Immunohistochemical stain for BMP-2at the14th day after cell transplantation:no significant difference between3days group and7days group, but more than that in no differentiation group and14days group; BMP-2positive cells were significantly more in local injection group than that in venous injection group; BMP-2positive cells were more in experimental group than that in control group.Conclusion1. Local injection could promote homing of hUC-MSCs compared with venous injection, and the homing of hUC-MSCs is not related with osteogenic induction in vitro.2. Osteogenic induction is good for survival of hUC-MSCs;3days group and7days group are more effective than14days group.3. Transplantation of hUC-MSCs could enhance the expression of BMP-2in bone injury area and osteogenic induction could enhance this phenomenon.4. BMP-2expression intensity in bone injury area:3days and7days group>14days group>transplantation without osteogenic induction> no transplantation.5. Systemic inflammatory response can’t be controlled by hUC-MSCs transplantation in bone injury model rats. |
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