以綠螢光豬骨髓間葉幹細胞異體移植入紅螢光豬於骨生成之應用-於支架中比較宿主細胞與移植細胞

Abstract

細胞，支架，骨誘導因子及血流供應是骨再生醫學重要的組成，但我們始終對最後再生產物中，植入細胞與宿主細胞的比例及相互關係並不清楚。根據以往研究我們曾用同種異體骨髓幹細胞轉染質體植入小鼠頭蓋骨缺損，且成功誘導分化，但因再生產物植入細胞與宿主細胞無法分辨，因此仍無法解答兩種細胞在再生產物的腳色。因此，我們設計使用異體綠螢光豬骨髓間葉幹細胞當作細胞來源，植入紅螢光豬頭蓋骨缺損，來藉此觀察在骨缺損情況下，再生產物中兩種螢光細胞比例及分布情況。 本論文的首要部分，我們先在體外支架上植入不同密度之異體綠螢光豬骨髓間葉幹細胞，以電子顯微鏡與磷酸酶及茜素紅等染色發現高密度細胞組較地密度組有更強螢光表現與更多骨分化。緊接著探討是否骨分化會影響螢光表現，結果呈現自綠螢光豬分離出之骨髓間葉幹細胞在骨誘導28天內仍保留其螢光特性且螢光亦不影響骨生成。 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。 利用綠螢光豬骨髓間葉幹細胞移植入異體紅螢光豬，較大鼠實驗不僅可以排除個體差異及免疫反應，更能為組織工程中移植細胞占比及植入體中分布情況與宿主細胞交互作用得到完整解答，同時將此觀念應用至後續骨髓間葉幹細胞招募機轉。

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.