| Objectives:Based on the observation of the evolution process of various cells in the process of DDM inducing osteogenesis, the immunohistochemical identification of mesenchymal stem cells, osteoblasts and chondrocyte, and the observation of osteoclasts by TRAP staining. The aim is to explore the theoretical framework of cell and the forms of osteogenesis in the mechanism of DDM inducing osteogenesis.Materials and Methods:Twenty-four3-month-old-big-eared New Zealand white rabbit participated in this study. Four erector spinae bags was created in each animals, three of them implaned DDM50mg(wet weight, the same below) by random selection and the another implanted nothing. The rabbits were sacrificed after1,2,3,4,8,12,16and20weeks and the samples examined HE staining, TRAP staining and immunohistochemical staining to indentificate mesenchymal stem cells, osteoblasts, chondrocytes, and identification of osteoclasts. The Image-Pro Plus6.0Image analysis software was measured the integrated option density (IOD) of Immunohistochemistry, and analyzed mesenchymal stem cells, osteoblasts and chondrocytes. All the datas were used SPSS17.0statistical software to statistical analysis.Results:1The General ObservationAfter operation, all the animals were wide-awake, their mental state is well, activity, diet. Their wounds had no cracking, inflammation, bleeding and leak. On the third day, parts of their wounds healed up, some sutures were bitten off.A week later, their hair was long, some covered the healed wounds. All animals were in good condition, had normal diet and activity.When obtaining the samples, there were closer integration between DDM and muscle.All specimens were not seen no inflammation in the surrounding tissues of DDM.2Histological Observation(1) HE StainingIn the control group, the muscle fibers arranged in parallel and the cell nucleus that are fusiform shape located in the peripheral cytoplasm. From the first week to twenty week, there were no inflammatory cell among the muscles. In the experimental group: the first week, DDM had been attached by the surrounding tissue, and some had not tightly attached with gaps. The surrounding of DDM was came around by cells, and showed granulation tissue, small blood vessels, inflammatory cells and fibroblasts spindle cells, multinueleated giant cells. The second week, DDM had been closely attached, the number of surrounding tissue cells reduced, and visibled basophils. The third week, there formed some cable structures among the tissue of DDM, visibled some light-blue-bock cartilage-like matrix, homogeneous red-dye osteoid-like matrix and cubic-shape osteoblast-like cells. The fourth week, showed that homogeneous red-dye osteoid matrix located in the surrounding of DDM, which’s surface coated cube-shaped osteoblastic-like cells. The eighth week, the edge of the DDM particles re-mineralized, whiah was purple-blue, and visibled some cells adhered absorption edge. There formed osteoid-like matrix, which surface had cube-shaped osteoblast-like cells, and eosinophilic cells. The twelfth week and sixteenth week, the surrounding of DDM particles visibled vacuolar fat cell, nonporous homogeneous red-dye-block bone-like tissue. The edge of DDM had covered cube-shape osteoblast-like cells, blood vessels, in which visibled double concave-shape red blood cells. The twentieth week, there visibled cartilage-like matrix in the surrounding tissues of DDM, where coated osteoblast-like cells. DDM particles visibled re-mineralization, whiah was purple-blue, and peripheral layer of DDM formed a film structure by fibroblasts.(2) TRAP Staining and Immunohistochemical Staining1) CD44Immunohistochemical StainingIn the experimental groups, CD44positive expression visibled around the surrounding tissues of DDM, not clingy DDM in the first week, then gradually close to DDM. The amount of expression enhanced gradually, peaked until the fourth week. From the fourth to twentieth week, positive expression dropped off. This indicated that mesenchymal stem cells increased gradually and then decreased. At the same time, MSCs gathered from the surrounding tissues to DDM. Analysis of variance:the first week with the second, third, fourth, eighth, twelfth week, the second week with the first, third, fourth, twetietf week, the third week with the first, second, fourth, eighth, sixeenth and twentieth week, the fourth week with each time, between fourth and sixeenth, twentieth week, the difference was statistically significant (P<0.05). While the control group had no positive expression.2) ALP Immunohistochemical StainingALP positive expression in the experimental group were closer to DDM, and gradually expanded to the periphery. The number of ALP expression accumulated from the first week, peaked until the fourth week, and after the fourth week, positive expression gradually reduced. Tt demonstated that the osteoblast begun by DDM edge, and spreaded gradually to the peripheral and matured. The fourth week later, the number of osteoblast began to gradually reduce. Analysis of variance:in the first, third, fourth and eighth week, in the second, third, fourth, sixeenth and twentieth week, in the third week with each time, among eighth week, sixteenth, twenieth week, the difference was statistically significant (P<0.05). As well as the control group had no positive expression.3) Immunohistochemical Staining of Collagen ⅡThe positive expression of collagen Ⅱ around DDM appeared and escalated from the first week of the experimental group, and slightly reducd in the fourth week. It reached its peak on the eighth week,from now on died down but no significant decrease. Analysis of variance:the first week with other various time points, between the second week and the eighth week, the fourth week and eighth week, the eighth week and twentieth week, the difference was statistically significant (P<0.05). Moreover there was no expression in the control group.4) TRAP Staining (tartrate-resistant acid phosphatase Staining)The positive control group showd that the enzymic active site presented reddish orange, located in the cytoplasm, and the nucleus is negative. The control and experimental groups showed no expression. It indicated that had no osteoclastogenesis in this period of the experimental observation.Coclusions:1After DDM had been implanted in the rabbit erector spinae bags, there has no tissue inflammation, Immunological rejection and necrosis in the observative period.This demonstates that DDM has well histocompatibility.2Histological observation:there appears chondroid-like matrix, osteoid matrix, cartilage-like cells and osteoblast-like cells in the surrounding of DDM. It makes clear that experimental animal model of the ectopic osteogenesis of DDM is successful, it proves that DDM has osteoinduction at the same time.3From the period of experimental observation, regardless of the amount of the expression of collagen Ⅱ in cartilage-like cells or its’ duration, more and longer than the expression of ALP in osteoblast-like cells. This indicates that DDM induced ectopic osteogenesis mode is mainly endochondral bone, whether intramembranous bone needs further study.4In the period of the experiment observation, no osteoclasts appear or generate by TRAP staining. |
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