含磷酸鍶之溫感性水膠誘導人類乳牙幹細胞骨分化研究

Stem Cells from Human Exfoliated Deciduous Teeth Differentiate into Osteoblasts by Thermo- Sensitive Hydrogel Including Strontium Phosphate

周瑋玲(wei ling chou)

指導教授 ： 蘇文達

國立臺北科技大學/工程學院/生化與生醫工程研究所/碩士(2014年)

https://doi.org/10.6841/NTUT.2014.00243

摘要

間葉幹細胞具有非常大的增殖潛力，只要少許細胞即可在體外增殖到足夠的數量。乳牙幹細胞是從幼兒牙髓中分離出的間葉幹細胞，這些細胞具有多向分化的能力，可分化為成骨細胞、軟骨細胞、脂肪細胞、肌肉細胞與神經細胞，能作為未來再生醫學的細胞來源。間葉幹細胞能分化成骨細胞的現象，已在許多研究中證實。本研究中以幾丁聚醣及β-磷酸甘油所組成的溫感性水膠，結合磷酸三鈣或磷酸鍶，形成3D網狀結構，探討磷酸鍶對乳牙幹細胞誘導骨分化的能力，並與傳統磷酸三鈣及單純水膠做比較。
製作好的水膠經流變儀測試及掃描式電子顯微鏡分析(Scanning electron microscopy)測定材料的基本物性。將乳牙幹細胞培養於本實驗室所製成的水膠中，培養1、5、7、14天，結果發現乳牙幹細胞在水膠中貼附性良好。經MTT測試後也證實此材料無明顯的細胞毒性。持續觀察21天後，含磷酸鍶水膠之ALP活性及鈣化沉積明顯高於另外兩組水膠。培養7天後有骨分化早期相關基因及BMP-2蛋白的表現，第14天時可觀察到CLO-1、RUNX2在含鍶水膠上相對於對照組有8.24及5.26倍的相對表現量，此含鍶水膠之表現量相較於另外兩組水膠高約2倍。在第21天時明顯表現較晚期的骨相關基因OCN及OPN，而在三組水膠中仍以含鍶水膠表現量最高。以上結果顯示，溫感性水膠結合磷酸鍶能有效促進乳牙幹細胞分化成骨細胞，未來可應用在骨組織工程上。

關鍵字

磷酸鍶 ； 磷酸鈣 ； 乳牙幹細胞 ； 骨分化 ； 水膠

並列摘要

The application and manipulation of mesenchymal stem cells (MSCs) in tissue engineering is a novel treatment modality in a great variety of pathologies. They include a high quality repair with regeneration of the injured tissue but without fibrous tissue formation. Furthermore, MSCs have high proliferation potential, only a small number of cells are required with subsequent expansion ex-vivo prior implantation. Stem cells from human exfoliated deciduous teeth (SHEDs) had been isolated from the human exfoliated deciduous teeth. They exhibit multilineage differentiation capacity being capable to give rise to diverse cells like osteoblasts, chondrocytes, adipocytes, myocytes and possibly neural cells. There are many studies indicated that MSCs can differentiation into osteocytes. In this study, we hope that SHEDs can proliferation, differentiation and calcification into osteoblasts in 3D scaffold that is porous thermo- sensitive hydrogel including tricalcium phosphate or strontium phosphate with chitosan and β-glycerophosphate for cellular growth.
The used characteristic of fabricated scaffolds was measured by the meter of rheology and Scanning electron microscopy. After, SHEDs were seeded into the hydrogel, cell viability were measured by MTT assay for 1, 5 ,7 and 14 days respectively. The results appeared the cellular attachment well and no apparent cytotoxicity for fabricated hydrogels was found for SHEDs. After sustained culture to 21 days, there are obvious ALP activity and calcium deposits in containing strontium phosphate hydrogel was significantly higher than the other two groups. The expression of BMP-2 protein was observed at 7 days culture in all hydrogels.In addition, significantly increased expression of COL-1 (8.24 times) and RUNX2 (5.26 times) were in 14 days culture in strontium phosphate- containing hydrogel group .
To sum up, thermo- sensitive hydrogels can enhance the osteogenic. differentiation of human exfoliated deciduous teeth stem cells, and thus can be a powerful utilization in tissue engineering in the future.

並列關鍵字

Strontium Phosphat ； Stem Cells from Human Exfoliated Deciduous Teeth ； Osteo-differentiation, ； hydrogel

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