Department of Neurosurgery, Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, No. 61, West Jiefang Road, Furong District, Changsha, 410005, Hunan, People's Republic of China.
Research and Development Center, Hunan Chuang He Biotechnology Limited Company, Changsha, 410205, Hunan, People's Republic of China.
Cell Tissue Bank. 2024 Sep;25(3):755-764. doi: 10.1007/s10561-024-10142-3. Epub 2024 Jul 8.
Cryopreservation is a method adopted for storage of autologous skulls. Herein, this current research sought to explore the effects of different cryoprotectants on the biological characteristics of rat calvarial osteoblasts after cryopreservation. Neonatal Sprague-Dawley rats were selected and their skull tissues were isolated. The skull tissues were allocated into the refrigerating-3M, refrigerating-6M, M199-3M, M199-6M, povidone iodine-3M, and povidone iodine-6M groups according to the usage of cryoprotectants and treatment time (month) and the fresh group. Osteoblasts were isolated from skull tissues in each group through digestion. The histomorphology of the skull was evaluated by H&E staining and cell morphology was observed by microscopy. The viability, proliferation, apoptosis, and osteogenic activity of osteoblasts were assessed by trypan blue staining, MTT, flow cytometry, and alkaline phosphatase (ALP) staining. The skull histomorphology and osteoblast morphology were similar between the fresh and refrigerating groups. Osteoblast viability was weakened after cryopreservation. The longer the refrigeration time, the lower the number of living cells and the higher the apoptosis rate. However, cryopreservation using different cryoprotectants did not evidently affect osteoblast proliferation and ALP activity. Different cryoprotectants show no apparent effect on the osteogenic activity of rat calvarial osteoblasts after cryopreservation.
冷冻保存是储存自体颅骨的一种方法。本研究旨在探讨不同冷冻保护剂对冷冻保存后大鼠颅骨成骨细胞生物学特性的影响。选用新生 Sprague-Dawley 大鼠，分离其颅骨组织。根据冷冻保护剂的使用情况和处理时间（月），将颅骨组织分为冷藏-3M、冷藏-6M、M199-3M、M199-6M、聚维酮碘-3M 和聚维酮碘-6M 组，以及新鲜组。通过消化从每组颅骨组织中分离出成骨细胞。通过 H&E 染色评估颅骨的组织形态学，通过显微镜观察细胞形态。通过台盼蓝染色、MTT、流式细胞术和碱性磷酸酶（ALP）染色评估成骨细胞的活力、增殖、凋亡和成骨活性。新鲜组和冷藏组的颅骨组织形态学和成骨细胞形态学相似。冷冻保存后成骨细胞活力减弱。冷藏时间越长，活细胞数量越少，凋亡率越高。然而，使用不同的冷冻保护剂冷冻保存对成骨细胞的增殖和 ALP 活性没有明显影响。不同的冷冻保护剂对冷冻保存后大鼠颅骨成骨细胞的成骨活性没有明显影响。
