Fahmy M D, Almansoori K A, Laouar L, Prasad V, McGann L E, Elliott J A W, Jomha N M
Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada.
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada.
Cryobiology. 2014 Feb;68(1):50-6. doi: 10.1016/j.cryobiol.2013.11.006. Epub 2013 Nov 20.
Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose-injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy μm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me(2)SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose-injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me(2)SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me(2)SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.
关节软骨（AC）的玻璃化可以提高组织可用性，但需要高浓度的冷冻保护剂（CPA）。本研究调查了常用CPA造成的相对损伤。我们假设，五种常用CPA的原位软骨细胞剂量-损伤关系是非线性的，并且可以通过比较在浓度增加时暴露后的细胞死亡来确定相对损伤。使用了四名接受全膝关节置换手术患者的人AC样本。将70μm厚的切片按照逐步方案暴露于5种CPA（最大浓度 = 8 M）的递增浓度下；二甲基亚砜（Me(2)SO）、甘油（Gly）、丙二醇（PG）、乙二醇（EG）和甲酰胺（FM）。通过膜完整性染色来确定软骨细胞活力。统计分析包括t检验和非线性最小二乘法估计方法。发现所有CPA的软骨细胞剂量-损伤关系都是非线性的（S形最佳拟合）。对于本研究中的特定加载方案，数据确定了以下CPA浓度，在这些浓度下软骨细胞的恢复在统计学上与对照恢复有显著差异；Gly为1 M，FM和PG为4 M，Me(2)SO为6 M，EG为7 M。个体均值的比较表明，Gly暴露导致的恢复率最低，其次是PG，然后是Me(2)SO、FM和EG，顺序不特定。本研究的信息提供了常用CPA对用于AC玻璃化的人原位软骨细胞造成损伤的顺序，并确定了逐步加载方案中软骨细胞恢复显著降低的CPA阈值浓度。一般来说，Gly和PG的损伤最大，而DMSO和EG的损伤最小。
