Mase Junji, Mizuno Hirokazu, Okada Kunihiko, Sakai Kensuke, Mizuno Daiki, Usami Kazutada, Kagami Hideaki, Ueda Minoru
Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Japan.
Cryobiology. 2006 Apr;52(2):182-92. doi: 10.1016/j.cryobiol.2005.10.013. Epub 2005 Dec 19.
Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.
越来越多的证据表明，骨膜细胞具有很大的骨再生潜力。我们已经证明，膜状培养骨膜（CP）是一种有效的牙槽骨再生装置。为了在临床环境中提高CP的可用性，已经开发了一种有效的CP冷冻保存方案。在本研究中，使用了三种不同的冷冻保护剂（二甲基亚砜、甘油和乙二醇）。研究了预孵育温度、预孵育时间和孵育过程中的搅拌对细胞活力的影响。样品在-196℃下储存10天。通过使用四唑盐的比色细胞活力测定法评估细胞活力，并用钙黄绿素AM和乙锭同二聚体-1组合染色后，通过共聚焦激光扫描显微镜确认测定结果。通过碱性磷酸酶测定法评估解冻后细胞的活性。为了评估冷冻保存的CP的成骨潜力，将CP移植到无胸腺大鼠的颅骨缺损处。在37℃下用二甲基亚砜平衡30分钟、搅拌的组中获得了最大的细胞活力，回收率为63.3±10.5%。冷冻保存后，当使用二甲基亚砜作为冷冻保护剂时，存活细胞的生长情况相同。用二甲基亚砜和甘油冷冻保存的组中碱性磷酸酶（ALP）活性得以维持。移植实验表明，通过移植冷冻保存的CP，颅骨缺损完全闭合，这表明CP的成骨特性得到了很好的维持。已经开发出一种有效的CP冷冻保存方案，这将为临床骨再生提供一种方便有效的治疗选择。
