骨髓間質細胞球體於3D列印聚乳酸支架之生長與成骨分化

Growth and Osteogenic Differentiation of Bone Marrow Mesenchymal Cell Spheroids in 3D Printed Polylactic Acid Scaffolds

呂秉廷(Ping-Ting Lu)

指導教授 ： 陳羿貞

共同指導教授 ： 鄭景暉(Jiiang-Huei Jeng)

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101092

摘要

前言
3D列印細胞支架是現今組織工程熱門的研究主題之一，3D環境進行細胞培養與在2D平面培養細胞，細胞的表現會有所不同，除了3D細胞支架以外，促使細胞聚集成球也是常見的3D細胞培養方式。本研究的目的是探討骨髓間葉細胞球體在3D列印細胞支架中的生長及成骨分化效果，以評估將此生長模型應用在骨組織工程的可能性。
材料與方法
聚乳酸(PLA)細胞支架以ultimaker 2+經由熱堆疊方式製作，製作成孔徑200 μm，層數為五層的3D細胞支架。再透過鹼化合併聚多巴胺進行表面處理，並針對表面處理後的細胞支架進行拉伸檢測。後續以Abcam的永生化人類骨髓間質細胞進行2D培養與3D細胞球體培養後，將分散細胞與球體細胞，分別進行細胞生長與成骨分化測試，並藉由西方墨點法研究2D培養細胞與3D細胞球體的蛋白質表現差異。此外也利用共軛焦顯微鏡進行3D細胞支架中細胞生長分析。
結果與討論
為了解3D列印PLA支架在經過表面處理後的楊氏模數的變化，我們利用拉伸測試看平面PLA圓盤跟3D PLA細胞支架的物理性改變。在平面圓盤的部分，表面處理會弱化其抗拉伸強度，但是3D PLA支架並無顯著差異，在孔隙率的分析也顯示表面處理微幅提升3D支架的孔隙率，但是並不具有統計上的顯著性。在2D細胞培養與3D細胞球體培養下的西方墨點分析顯示: 隨著細胞成球的天數上升，HIF-1α、CD44和VEGF表現下降，而CD73表現則無明顯變化。
在細胞生長測試方面，使用聚多巴胺合併鹼化表面處理後的3D列印PLA支架有利於3D細胞球體的貼附和生長。在2D培養細胞的組別，經過表面處理的3D細胞支架反而不利於初期細胞生長。以共軛焦顯微鏡觀察，我們可以發現3D細胞球體在表面處理後的3D列印PLA支架中，細胞與支架的貼附效果優於其他組別。
在成骨分化的分析，包括鹼性磷酸酶(ALP)活性測試，與茜素紅S(ARS)染色及定量分析細胞外基質礦化程度，由ALP的測試可知TCPS上3D細胞球體的ALP活性高於2D培養細胞。ARS染色結果也發現3D細胞球體在表面處理後的3D PLA支架中表現的成骨分化結果優於其他組別。
結論
結果顯示3D細胞球體在表面處理過的3D細胞支架中的細胞生長及成骨分化優於2D培養細胞，並且在這種模型下iBMC細胞球體對於支架的貼附性也優於2D培養細胞，此模式或許有利於成骨組織工程研究。

關鍵字

細胞成球 ； 3D列印聚乳酸支架 ； 聚多巴胺表面處理 ； 成骨分化

並列摘要

Introduction
3D printing scaffolds is one of the popular research of tissue engineering. Many studies have pointed out that the results of cell culture in a 3D environment and cells in 2D condition will be different. In addition to 3D cell scaffolds, aggregation cell culture is a common 3D cell culture method. Studies on osteogenic differentiation have confirmed that these two culture methods have the potential to improve osteogenic differentiation. Therefore, the purpose of this study is to realize the effect of osteogenic differentiation with aggregation cell culture combined with 3D printed cell scaffolds.
Materials and Methods
The 3D Polylactic acid (PLA) cell scaffolds are all made by ultimaker 2+ through fused deposition modeling, the pore size was 200 μm with 5 layer and use alkalization and polydopamine for surface treatment. The tensile physical properties of the cell scaffold after surface treatment are tested. For cell research, Abcam’s immortalized human bone marrow mesenchymal cells were used for cell growth and osteogenic differentiation research with 2D cell culture and 3D cell spheroids. Western blotting was used for the difference in protein expression between 2D cell culture and 3D cell spheroids. In addition, confocal microscopy was used for analysis of cell growth in 3D scaffold.
Results and discussion
In order to understand the physical properties of the 3D printing scaffold after surface treatment, we used a tensile test to see the Young’s modulous of the PLA disc and the 3D PLA cell scaffold. As a result, it seems that part of the surface treatment on the disc will weaken the physical properties, but there will be no significant difference in the physical properties of the 3D PLA scaffold. The porosity study also shows that the surface treatment will increase the porosity of the 3D scaffold but it is not statistically significant.
Western blot analysis showed that as the days of the aggregation cell culture, the expression of HIF-1α, CD44 and VEGF were decreased, while the expression of CD73 did not change significantly.
In the cell growth test, it can be found that polydopamine and alkalized surface treatment of 3D cell scaffolds is beneficial to the attachment and growth of 3D cell spheroids. However, for 2D cultured cells, the surface treatment 3D cell scaffold is not favor to the initial cell growth. Under the results of the confocal microscope, we can find that the 3D cell spheroids and the surface treatment 3D cell scaffold have a better cell attachment to scaffold than other combinations.
In osteogenic differentiation, the alkaline phosphatase (ALP) activity of cell spheroid cultured on TCPS is better than single cells. In the results of alizarin red S staining (ARS), the results of cell spheroid and 3D cell scaffolds with surface treatment are better than other groups.
Conclusion
The results show that the cell spheroid with surface treatment 3D cell scaffold is better than that of 2D cultured cells. In this model, the adhesion of iBMC to the scaffold is also better than that of 2D cultured cells. It may be a favorable model in osteogenic tissue engineering.

並列關鍵字

cell spheroid ； 3D printing polylactic acid scaffold ； polydopamine surface treatment ； osteogenic differentiation

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