Li Mengying, Feng Cheng, Gu Xiuge, He Qin, Wei Fulan
Department of Orthodontics, School of Stomatology, Shandong University, Jinan, People's Republic of China.
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Wenhua Xi Road No. 44-1, Jinan, Shandong, 250012, People's Republic of China.
Stem Cell Res Ther. 2017 Apr 17;8(1):77. doi: 10.1186/s13287-017-0530-5.
Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets.
PDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration.
The PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix.
Our results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets and could enhance their clinical utility in tissue regeneration.
冷冻保存已广泛应用于多种生物材料的长期储存。然而，目前尚无关于牙周膜干细胞（PDLSC）片层冷冻保存的全面研究，而PDLSC片层被认为是牙周组织再生的优良移植材料。本研究旨在探讨冷冻保存对PDLSC片层结构完整性和功能活性的影响。
从拔除的人类磨牙制备的PDLSC片层分为两组：冷冻保存组（cPDLSC片层）和新鲜制备的对照组（fPDLSC片层）。cPDLSC片层在由90%胎牛血清和10%二甲基亚砜组成的溶液中冷冻保存3个月。分别通过细胞活力测定和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定评估两组中PDLSC的细胞活力和细胞增殖率。通过冯·科萨染色和油红O染色评估细胞的多向分化潜能。通过核型分析检查染色体稳定性。此外，将每组的细胞片层皮下移植到裸鼠的背部，之后进行天狼星红染色以分析组织再生效率。
两组细胞片层来源的PDLSC在活力、增殖能力、多向分化潜能以及染色体稳定性方面均无显著差异。此外，基于小鼠模型的移植实验表明，cPDLSC片层在体内生成有活力的类骨质组织方面与新鲜制备的对应物同样有效。在两种情况下，再生组织均显示出类似的骨样基质网络模式。
我们的结果提供了令人信服的证据，即冷冻保存不会改变PDLSC片层的生物学特性，并且可以提高其在组织再生中的临床应用价值。
