磁性冷凍對牙髓幹細胞數量與特性之影響

Influences of Magnetic Cryopreservation On the Dental Pulp Stem Cells

蔣寶漳(Pao-Chang Chiang)

指導教授 ： 李勝揚

共同指導教授 ： 黃豪銘

臺北醫學大學/口腔醫學院/牙醫學系碩博士班/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00035

摘要

研究背景及目的：近年來，科學界已研發出能夠成功保存牙周韌帶的特殊磁性冷凍法，可在降溫中形成玻璃化而避免因冰晶產生所造成的細胞傷害。藉由此技術可保存因齒顎矯正需求或感染預防而拔下的健康牙齒，作為未來自體再植。而隨著幹細胞科技發展，人類牙髓間葉幹細胞已被成功培養並鑑定，有機會被應用於再生醫療。本研究旨在擴大磁性冷凍的應用範圍，讓此一技術不只是能完整冷凍保存牙周韌帶以供解凍後再植，更能長期保存齒內牙髓組織，以供未來解凍後其中間葉幹細胞之分離與應用。

研究材料及方法：在牙齒銀行中保存的牙齒設定為程式降溫冷凍組，同一患者之對側牙齒不予冷凍，設為未冷凍組。另外，並設計傳統冰箱-20 ℃冷凍組（以-20 ℃冰箱取代磁性程式降溫儀）。三組牙齒皆以組織塊酶解法培養出牙髓幹細胞。首先評估三組牙髓幹細胞之培養成功率（以未冷凍組作為標準，評估程式降溫冷凍組與傳統冰箱-20 ℃冷凍組是否受到冷凍影響），進而比較分析程式降溫冷凍組與未冷凍組之生長曲線（以MTT 法），形態差異（掃描式電子顯微鏡影像），表面特徵蛋白（CD44與STRO-1之免疫螢光染色）以及分化能力（脂肪化與骨化）。

結果：程式降溫冷凍組有73 ％的牙齒並未受到冷凍影響，仍然能夠成功培養牙髓幹細胞，而傳統冰箱-20 ℃則僅有20 %的牙齒能夠成功培養牙髓幹細胞。以MTT法評估程式降溫冷凍組與未冷凍組之生長曲線無統計上顯著差異（p < 0.05）；電子顯微鏡之影像在程式降溫冷凍組與未冷凍組間亦無明顯差異；程式降溫冷凍組與未冷凍組之牙髓幹細胞也都能表現CD44和 STRO-1兩種表面特徵蛋白以及脂肪化與骨化的能力。

結論：本研究結果顯示磁性冷凍可將齒內牙髓組織以整顆牙齒的方式進行程式降溫冷凍保存，並培養出同樣生長潛力與表面特徵蛋白之牙髓間葉幹細胞，也能同樣表現多元分化能力。牙科患者未來儲存牙齒在程式降溫冷凍牙齒銀行內，不僅可供未來解凍後再植，亦可望視需要而培養其中牙髓幹細胞以提供臨床利用。

關鍵字

牙髓幹細胞 ； 冷凍保存 ； 牙齒銀行 ； 再生醫療 ； 多元分化

並列摘要

Background & Objectives: Recently, scientists have successfully cryopreserved the periodontal ligament with a special magnetic cryopreservation method, which could induce vitrification to avoid the injuries from ice crystals to the cells. Consequently, the healthy teeth extracted for orthodontic treatment or prevention of infection could be potential donor teeth for future auto-transplantation. With the advancement of stem cell technology, human dental pulp stem cells have been cultured and could be used in regenerative medicine. This study was aiming to expand the application of Tooth Bank from cryopreservation for auto-transplantation to long-term storage of dental pulp stem cells.

Materials & Methods: The teeth stored in Tooth Bank were designed as the programming cryopreserved group. The non-cryopreserved group was teeth on contralateral side of the same patient. Besides, there was a traditionally freezing group (cryopreserved with -20 ℃ traditional freezer instead of program freezer with magnetic field used in the programming cryopreserved group). Dental pulp stem cells were isolated with enzyme digestion method from these 3 groups. First, the successful rate of culturing dental pulp stem cells in these 3 groups would be evaluated (by the standard set in non-cryopreserved group). Furthermore, the growth curve (by MTT method), morphology (by scanning electronic microscope, SEM), surface markers of stem cells (by immunostaining of CD44 and STRO-1) and ability of differentiation (adipogenic and osteogenic differentiation) of the dental pulp stem cells in the programming cryopreserved group and the non-cryopreserved group were evaluated and compared.

Results: The dental pulp stem cells in 73 % of the teeth in the programming cryopreserved group were considered as not been influenced after cryopreservation, since they could be normally cultured. However, the dental pulp stem cells in only 20 % of the teeth in the traditionally freezing group could be normally cultured. The growth curves evaluated by MTT method had no statistical differences between programming cryopreserved and non-cryopreserved groups. The morphology observed from SEM showed similar variety of cells in both groups. Both of dental pulp stem cells isolated from the programming cryopreserved group and the non-cryopreserved groups showed positive surface markers of stem cells (CD44+ and STRO-1+) and ability of adipogenic and osteogenic differentiation.

Conclusion: The results of this study indicated that dental pulp stem cells would remain their growth potential, surface markers and multi-lineage differentiation ability even after magnetic programming cryopreservation of the whole teeth. Patients who store teeth in Tooth Bank will have teeth not only for auto-transplantation but also have the chance of later isolation of dental pulp stem cells in the future needs.

並列關鍵字

Dental pulp stem cells ； Cryopreservation ； Tooth bank ； Regenerative medicine ； Multi-lineage differentiation

參考文獻

Almushayt, A., K. Narayanan, A. E. Zaki, and A. George. 2006. Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Ther 13 (7):611-620.

Andreasen, J. O., H. U. Paulsen, Z. Yu, T. Bayer, and O. Schwartz. 1990. A long-term study of 370 autotransplanted premolars. Part II. Tooth survival and pulp healing subsequent to transplantation. Eur J Orthod 12 (1):14-24.

Arthur, A., G. Rychkov, S. Shi, S. A. Koblar, and S. Gronthos. 2008. Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26 (7):1787-1795.

Broxmeyer, H. E., E. F. Srour, G. Hangoc, S. Cooper, S. A. Anderson, and D. M. Bodine. 2003. High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years. Proc Natl Acad Sci U S A 100 (2):645-650.

Burbridge, L., B. O. Cole, R. S. Hobson, and R. R. Welbury. 2005. Auto-transplantation in the restorative management of traumatized anterior teeth: a case report. Dent Update 32 (9):529-530, 532-524.