Malekfar Azin, Valli Kusum S, Kanafi Mohammad Mahboob, Bhonde Ramesh R
Department of Conservative Dentistry and Endodontics, Sri Rajiv Gandhi College of Dental Sciences and Hospital, Bangalore, India.
Manipal Institute of Regenerative Medicine, Manipal University, Bangalore, India.
J Endod. 2016 Jan;42(1):76-81. doi: 10.1016/j.joen.2015.10.001. Epub 2015 Nov 11.
Human dental pulp stem cells (DPSCs) are becoming an attractive target for therapeutic purposes because of their neural crest origin and propensity. Although DPSCs can be successfully cryopreserved, there are hardly any reports on cryopreservation of dental pulp tissues obtained from teeth diagnosed with symptomatic irreversible pulpitis during endodontic treatment and isolation and characterization of DPSCs from such cryopreserved pulp. The aim of this study was to cryopreserve the said pulp tissues to propagate and characterize isolated DPSCs.
A medium consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide was used for cryopreservation of pulp tissues. DPSCs were isolated from fresh and cryopreserved pulp tissues using an enzymatic method. Cell viability and proliferation were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. DPSC migration and interaction were analyzed with the wound healing assay. Mesenchymal characteristics of DPSCs were verified by flow cytometric analysis of cell surface CD markers. The osteogenic and adipogenic potential of DPSCs was shown by von Kossa and oil red O staining methods, respectively, and the polymerase chain reaction method.
We found no significant difference in CD marker expression and osteogenic and adipogenic differentiation potential of DPSCs obtained from fresh and cryopreserved dental pulp tissue.
Our study shows that dental pulp can be successfully cryopreserved without losing normal characteristics and differentiation potential of their DPSCs, thus making them suitable for dental banking and future therapeutic purposes.
人牙髓干细胞（DPSCs）因其神经嵴起源和特性，正成为治疗目的的一个有吸引力的靶点。尽管DPSCs能够成功冻存，但关于在牙髓治疗期间从诊断为有症状的不可逆性牙髓炎的牙齿中获取的牙髓组织的冻存，以及从这种冻存牙髓中分离和鉴定DPSCs的报道几乎没有。本研究的目的是冻存上述牙髓组织，以扩增和鉴定分离出的DPSCs。
使用由90%胎牛血清和10%二甲基亚砜组成的培养基冻存牙髓组织。采用酶法从新鲜和冻存的牙髓组织中分离DPSCs。使用MTT[3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐]法测定细胞活力和增殖。通过伤口愈合试验分析DPSC的迁移和相互作用。通过对细胞表面CD标志物的流式细胞术分析验证DPSCs的间充质特征。分别通过冯·科萨染色法和油红O染色法以及聚合酶链反应法显示DPSCs的成骨和成脂潜能。
我们发现从新鲜和冻存牙髓组织中获得的DPSCs在CD标志物表达以及成骨和成脂分化潜能方面没有显著差异。
我们的研究表明，牙髓能够成功冻存，而不会丧失其DPSCs的正常特征和分化潜能，从而使其适用于牙髓库保存和未来的治疗目的。
