Wang Lihong, Pegg David E, Lorrison Jonathan, Vaughan David, Rooney Paul
Medical Cryobiology Unit, Department of Biology, University of York, UK.
Cryobiology. 2007 Oct;55(2):138-47. doi: 10.1016/j.cryobiol.2007.06.005. Epub 2007 Jul 4.
The cryopreservation of articular cartilage with survival of living cells has been a difficult problem. We have provided evidence that this is due to the formation of ice crystals in the chondrons. We have developed a method in which the concentration of the cryoprotectant dimethyl sulphoxide (Me(2)SO) is increased progressively, in steps, as cooling proceeds so that ice is never allowed to form, but the very high concentrations of Me(2)SO required at low temperatures are reached only at those low temperatures. In this paper, we describe some new experiments with discs of ovine articular cartilage similar to those used in our previous studies and we show that continuous stirring throughout the process resulted in a significant increase in the rate of (35)S sulphate incorporation into glycosoaminoglycans (GAGs), now reaching 87% of the corresponding fresh control values. We confirmed that the method is also effective for human knee joint cartilage, which gave 70% of fresh control ability to synthesise GAGs; continuous stirring was also used in this experiment. We then extended the method to ovine knee joint osteochondral dowels and showed that, again with continuous stirring, the method produced tissue concentrations of Me(2)SO that were sufficient to prevent freezing in dowels too, and to permit cell function at 60% of control. The most important mechanical property (instantaneous compressive modulus) was unaffected by the process. Finally, we experimented with some technical variations to facilitate clinical use-a more rapid process for warming and removal of Me(2)SO was developed and a method of short-term storage before or after cryopreservation was developed. Finally, pilot experiments were carried out to provide proof of principle for a closed, continuous flow method in which both temperature and Me(2)SO concentration were computer-controlled.
在保证活细胞存活的前提下对关节软骨进行冷冻保存一直是个难题。我们已证实这是由于软骨细胞中形成了冰晶所致。我们研发了一种方法，在降温过程中，冷冻保护剂二甲基亚砜（Me(2)SO）的浓度逐步递增，从而防止冰晶形成，且只有在低温时才会达到所需的极高浓度。在本文中，我们描述了一些用绵羊关节软骨圆盘进行的新实验，这些圆盘与我们之前研究中使用的类似，我们发现整个过程中持续搅拌可使(35)S硫酸盐掺入糖胺聚糖（GAGs）的速率显著提高，目前已达到相应新鲜对照值的87%。我们证实该方法对人膝关节软骨同样有效，其合成GAGs的能力达到新鲜对照的70%；本实验中也采用了持续搅拌。然后我们将该方法扩展至绵羊膝关节骨软骨柱，结果表明，同样在持续搅拌的情况下，该方法产生的Me(2)SO组织浓度足以防止骨软骨柱冻结，并使细胞功能达到对照的60%。最重要的力学性能（瞬时压缩模量）不受该过程影响。最后，我们对一些技术变体进行了实验以方便临床应用——开发了一种更快的升温及去除Me(2)SO的方法，并开发了一种在冷冻保存前后进行短期储存的方法。最后，进行了初步实验以验证一种温度和Me(2)SO浓度均由计算机控制的封闭连续流方法的原理。
