Detection Of Angiogenesis After Stem Cell Transplantation In A Rat Model Of Hindlimb Ischemia Using Synchrotron Radiation

Part One Detection of Microvasculature in Rat Hind Limb Using Synchrotron RadiationObjective:Conventional radiography cannot provide sufficiently clear images of vessels with diameters of 200?m or less. New X-ray microangiography and third-generation synchrotron radiation-based microcomputed tomography have opened new perspectives for microvascular imaging of extremity. Here we aimed to visualize deep-level microvascular structure in rat hind limb by microangiographic technique, and compare images with those by conventional method.Materials and Methods:Inbred strain rats (F344) were used for in vivo and ex vivo study. Microangiography in vivo and ex vivo was performed and images were compared with those by conventional method. In microangiography in vivo study, the catheter was connected to an automated angiographic injector, through which the nonionic contrast media was injected into blood vessels in rat hind limbs. A monochromatic 33.4 keV X-ray source just above the K-absorption edge of iodine was used for imaging. In vitro study, normal saline and barium sulfate solution was infused through catheter to the left iliac artery of rats, and the phase contrast imaging were performed with the energy of 15 Kev and the object-to-image distance of 800mm. Synchrotron radiation-based micro-computed tomography (SRmCT) was also performed to reveal three-dimensional (3D) morphology of the blood vessel in rat hind limb.Results:Using microangiographic technique in vivo and in vitro (with barium sulfate), blood vessels in the rat limb muscle could be visualized with high resolution and more detail compared with those by conventional technique, and the fifth to sixth branches of iliac artery in rat hind limb could be detected with the minimum visualized blood vessels about 40?m and 9?m in diameter, respectively. Accordingly, higher angiographic scores were achieved than those by conventional X-ray. In addition, the vascular network could be defined and analyzed at the micrometer scale from the 3D renderings of limb vessel as shown by SRmCT.Conclusions:Synchrotron radiation-based microangiography and SRmCT thus provided a practical and effective means to observe the microvasculature of rat hindlimb, which might be useful in assessment of angiogenesis in lower limbs. Part TwoThe study of culturing and multilineage potential of rat marrow-derived mesenchymal stem cellsObjective:Bone marrow mesenchymal stem cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in limb ischemic disease. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of plating density and culturing method on proliferation, cell morphology, and differentiation potential of MSC, and to provide an effective approach of cell obtaining for stem cell transplantiation.Materials and Methods:Rat MSCs were isolated from rat bone marrow relying on their ability to adhere to plastic, and replated at appropriate seeding density (3x10, 3x102 and 3x103 cells/cm2) at each passage. Cell morphology and growth characteristic were evaluated and compared among different seeding density. Passage 3 MSCs from cultures seeded at optimal plated density were used for identification of their surface marker by flow cytometry and immunofluorescence. For differentiation assays, cells from passage 3 were cultured in induction medium supplemented with different chemical agents and cytokines. Differentiation of MSCs into adipogenic, osteogenic, and endothelial lineages were identified by Oil Red-O stain for lipid deposits, stained for kaline phosphatase by an indoxyltetrazolium method, and incubated in the endothelial differentiation medium containing Dil-Ac-LDL and stained with BS-1-lectin, respectively.Results:Primary cultur of rMSCs showed a spindle-like or flattened polygonal appearance with the characteristic of adherence to plastic. The low density culture consisted of a relatively homogenous monolayer of spindle-like cells while high density cultures appeared heterogeneous in which nonfibroblastic cells were present. Cell colonies were observed in low density culture with high population doubling speed. Flow cytometric analysis of the low density culture at passage 3 demonstrated to be positive for CD90, CD29, CD105, but negative for CD34, CD45. Under certain conditions, rMSCs of low density culture were capable of differentiating into multilineage cell types, including adipogenic cells which were positive for oil red-0 stain, osteogenic lineages with positive stain for kaline phosphatase by an indoxyltetrazolium method, and endothelial cells with positive result of both LDL-uptaking assay and BS-1-lectin stain.Conclusion:Based on the results, we concluded that adherent culturing method combined with the low density culture system is a more appropriate system to isolate and expand murine MSCs from marrow samples. With low density culture system, more purified mMSCs which express putative surface maker and have multiple differential potential could be produced. density (AMVD) was increased at 14 and 21 days in both group A and group B, and significant higher in group A compared with group B (0.54±0.036 vs 0.34±0.032 and 0.64±0.037 vs 0.49±0.027, P?0.05, for 14 days and 21 days, respectively). In addition, the microvascular pattern and arrangement was more regular in group A, when compare with group B at 21 days after transplantation. There were no significant changes of microvasculature in the sham operation group (group C). A significant improvement of blood flow perfusion and lower injury score was found in group A, when compared with group B since 7 days after cell transplantation. The results of microvascular density by microangiography ex vivo indicated there was a higher microvascular density in group A than in group B, which were consistent with the results of immunohistochemistry stain.Conclusion:SR based microangiography is an effective method for the detection of angiogenesis after stem cell transplantation in a rat model of hindlimb ischemia, which suggested a higher microvascular density and more regular branching pattern of neovascularization in rMSC transplantation group compared with PBS group.