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Bone regeneration in Ds-Red pig calvarial defect using allogenic transplantation of GFP pMSCs – a comparison of host cells and seeding cells in the scaffold
mesenchymal stem cell,bone regeneration,enhanced green fluorescent,DsRed pig,pig calvarial defect model,
|Publication Year :||2020|
在本論文的最後體內實驗部分，我們設計五組試驗模式(第一組:骨缺損未植入任何材料;第二組: 骨缺損僅植入支架; 第三組: 植入加骨誘導液之支架; 第四組: 植入含5 x 103個綠螢光豬骨髓間葉幹細胞之支架; 第五組: 植入含5 x 103個綠螢光豬骨髓間葉幹細胞之支架)在每一隻本土紅螢光豬頭蓋骨上鑿出七個骨缺損(第一組至第三組各一骨缺損; 第四組及第五組各兩個骨缺損)。本計畫共使用八頭本土紅螢光豬，每周犧牲兩頭，螢光顯微鏡顯示綠螢光於整個骨再生過程中皆存在且高密度組在第四周有更多螢光表現，紅螢光宿主細胞需支架上有更多空間才能招募進來且無植入細胞無法招募宿主細胞，組織切片及免疫螢光染色皆顯示更多植入細胞會有更好骨分化現象。
Cells, scaffolds, and factors are the triad of regenerative engineering; however, it is difficult to distinguish whether cells in the regenerative construct are from the seeded cells or host cells via the host blood supply. We performed a novel in vivo study to transplant enhanced green fluorescent pig mesenchymal stem cells (EGFP-pMSCs) into calvarial defect of DsRed pigs. The cell distribution and proportion were distinguished by the different fluorescent colors through the whole regenerative period. The first part of present project, we transplant EGFP-pMSCs into gelatin scaffolds and found that the increasing fluorescence expression and osteogenic profiles as increased loaded number of cells indicate biocompatibility and biodegradability of scaffold after differentiation. In order to clarify if the expression of green fluorescence alter the magnitude of osteogenic differentiation, we compared transgenic pig-derived eGFP MSCs and nonviral eGFP-transfected MSCs and found that the fluorescence expression wound not interfere with osteogenic differentiation after osteoinduction for 28 days.
In our in vivo study of this project, eight adult domestic Ds-Red pigs were treated with five modalities: empty defects without scaffold (group 1); defects ﬁlled only with scaffold (group 2); defects ﬁlled with osteoinduction medium-loaded scaffold (group 3); defects filled with 5 x 103 cells/scaffold (group 4); and defects filled with 5 x 104 cells/scaffold (group 5). The in vitro cell distribution, morphology, osteogenic differentiation, and ﬂuorescence images of groups 4 and 5 were analyzed. Two animals were sacriﬁced at 1, 2, 3, and 4 weeks after transplantation. The in vivo ﬂuorescence imaging and quantification data showed that EGFP-pMSCs were represented in the scaffolds in groups 4 and 5 throughout the whole regenerative period. A higher seeded cell density resulted in more sustained seeded cells in bone regeneration compared to a lower seeded cell density. Host cells were recruited by seeded cells if enough space was available in the scaffold. Host cells in groups 1 to 3 did not change from the 1st week to 4th week, which indicates that the scaffold without seeded cells cannot recruit host cells even when enough space is available for cell ingrowth. The histological and immunohistochemical data showed that more cells were involved in osteogenesis in scaffolds with seeded cells. Our in vivo results showed that more seeded cells recruit more host cells and that both cell types participate in osteogenesis. These results suggest that scaffolds without seeded cells may not be effective in bone transplantation.
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