Cryopreservation of Osteochondral Allografts: Dimethyl Sulfoxide Promotes Angiogenesis and Immune Tolerance in Mice

C. Wingenfeld, R. Egli, A. Hempfing, R. Ganz, M. Leunig
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引用次数: 61

Abstract

Background: Although transplantation of cryopreserved bone allografts has become a routine procedure in orthopaedic surgery, biological and immunological impairment remains an unsolved problem that causes clinical failures. Experimental and clinical evidence has indicated that bone grafts that are revascularized early remain viable and contribute to union at the recipient site. Unprotected cryopreservation, used in most bone banks to reduce graft antigenicity, is associated with complete loss of graft viability, potentially contributing to graft failure. The differences in the survival of various cell types during cryopreservation with use of dimethyl sulfoxide, particularly the increased sensitivity of leukocytes to fast freezing, has resulted in a new approach to modulate immunogenicity. On the basis of this concept, it was proposed that a reduction in the immune response and enhanced revascularization of osteochondral allografts could be achieved by rapid cryopreservation with dimethyl sulfoxide. To test this hypothesis, angiogenesis and immune tolerance were quantified in a murine model with use of intravital microscopy.Methods: Fresh osteochondral tissue and osteochondral tissue that had been cryopreserved with and without dimethyl sulfoxide was transplanted into dorsal skinfold chambers as isografts and as allografts in presensitized and nonsensitized recipient mice. To quantify angiogenesis, the onset of hemorrhages in the vicinity of the grafts and the revascularization of the grafts were determined by means of intravital fluorescence microscopy. To determine the recipient's intravascular immune response to the grafts, the leukocyte-endothelium interaction was assessed on the twelfth day after transplantation.Results: Nine of nine fresh isografts were revascularized at a mean (and standard deviation) of 57 ± 33 hours, eight of nine isografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 98 ± 50 hours, and zero of nine isografts that had been cryopreserved without dimethyl sulfoxide were revascularized. Seven of seven fresh allografts were revascularized at 53 ± 6 hours, and ten of ten allografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 82 ± 29 hours. However, signs of revascularization faded in four of the seven fresh allografts whereas reperfusion was maintained in the majority (seven) of the ten grafts frozen in the presence of dimethyl sulfoxide. Similar to the findings associated with unprotected frozen isografts, zero of ten unprotected frozen allografts were revascularized. None of the allografts that had been transplanted into presensitized recipients were revascularized, regardless of whether they had been implanted fresh (nine grafts) or had been implanted after protected (eight grafts) or unprotected (nine grafts) freezing. Quantification of the leukocyte-endothelium interaction revealed a reduction in the intravascular immune response to frozen allografts (both protected and unprotected) compared with fresh allografts.Conclusion: Osteochondral allografts that had been pretreated by cryopreservation with dimethyl sulfoxide demonstrated improved angiogenesis induction and enhanced immune tolerance compared with unprotected frozen grafts. A selective reduction in donor passenger leukocytes is the proposed mechanism underlying this phenomenon.Clinical Relevance: In the absence of presensitization, cryopreservation with dimethyl sulfoxide appears to reduce the immune response to allografts and to enhance their revascularization; in the presence of presensitization, alternatives to allograft transplantation should be considered since the allografts will be exposed to a deleterious immune response.
异体骨软骨移植物的低温保存:二甲基亚砜促进小鼠血管生成和免疫耐受
背景:尽管低温保存同种异体骨移植已成为骨科手术的常规手术,但生物和免疫损伤仍然是一个未解决的问题,导致临床失败。实验和临床证据表明,早期血运重建的骨移植物保持活力并有助于受体部位的愈合。大多数骨库使用无保护的低温保存来降低移植物抗原性,这与移植物活力的完全丧失有关,可能导致移植物失败。在使用二甲亚砜进行低温保存期间,不同类型细胞的存活率存在差异,特别是白细胞对快速冷冻的敏感性增加,这导致了一种调节免疫原性的新方法。基于这一概念,我们提出可以通过二甲亚砜快速低温保存来降低免疫反应并增强同种异体骨软骨移植物的血运重建。为了验证这一假设,使用活体显微镜对小鼠模型中的血管生成和免疫耐受进行了量化。方法:将新鲜骨软骨组织和冷冻保存的骨软骨组织分别作为同种异体和同种异体移植到致敏小鼠和非致敏小鼠的背侧皮襞腔内。为了量化血管生成,通过活体荧光显微镜测定移植物附近出血的发生和移植物的血运重建。为了确定受体对移植物的血管内免疫反应,在移植后第12天评估白细胞-内皮相互作用。结果:9个新鲜同种移植物中9个在平均(和标准差)57±33小时内血管重建,9个用二甲亚砜冷冻保存的同种移植物中8个在98±50小时内血管重建,9个不使用二甲亚砜冷冻保存的同种移植物中0个血管重建。7例新鲜同种异体移植物在53±6小时血运重建,10例用二甲亚砜冷冻保存的同种异体移植物在82±29小时血运重建。然而,7例新鲜同种异体移植物中有4例血管重建迹象消退,而在二甲亚砜存在下冷冻的10例移植物中,大多数(7例)维持再灌注。与未受保护的冷冻同种异体移植物相似,未受保护的冷冻同种异体移植物的血管重建率为零。移植到致敏受体的同种异体移植物,无论是新鲜移植(9个移植物),还是在保护(8个移植物)或未保护(9个移植物)冷冻后移植(8个移植物),均未出现血运重建。白细胞-内皮相互作用的定量显示,与新鲜同种异体移植物相比,冷冻同种异体移植物(包括保护和未保护的)的血管内免疫反应降低。结论:与未经保护的冷冻移植物相比,经二甲亚砜冷冻保存预处理的同种异体骨软骨移植物具有更好的血管生成诱导和增强的免疫耐受能力。供体乘客白细胞的选择性减少是这一现象的潜在机制。临床相关性:在没有现敏化的情况下,二甲基亚砜低温保存似乎可以降低对同种异体移植物的免疫反应,并增强其血运重建;由于同种异体移植物将暴露于有害的免疫反应,因此在存在现敏化的情况下,应考虑替代同种异体移植物移植。
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