腰椎间盘同种异体移植后终板的结构与功能重构研究

Fresh-frozen intervertebral disc allograft transplantation has been regarded as a new strategy to regenerate the whole degenerated disc with promising clinical outcomes. Nevertheless, mild degeneration of the allograft has been found in some cases after follow-up, which can be attributed to the loss of timely nutrient supply. It is well known that the normal intervertebral disc obtains most of the nutrients through the endplate pathway; but immediately after transplantation, the allograft is immersed in an ischemic environment until the endplate nutritional pathway is reestablished. Loss of nutrient supply will induce the death of disc cells, leading to the presence of intervertebral disc degeneration. Hence, in order to unravel the reestablishment of the endplate nutritional pathway, we will investigate the structural and functional remodeling process of the endplates after disc allografting. In this project, based on the goat model of lumbar intervertebral disc allograft transplantation, we will firstly investigate the reestablishment pattern of the endplate nutritional pathway using the dynamic-enhanced MRI. Then we will perform UTE-MRI, micro-CT scanning, histology and immunochemistry to investigate the structural changes of the bony and cartilaginous endplates. We will also conduct the SEM, EDX mapping and histological staining to observe the micro-structural integration of bony and cartilaginous endplates. Finally, the cells isolated from the cartilaginous endplates of the disc allograft will be exposed to the hypoxia and serum deprivation conditions; the apoptosis of the cartilaginous endplate cells will be evaluated by FACS (Annexin V/PI) and the HIF-1α-HMGB1 based mechanism will be determined by ELISA, PCR and Western Blot studies. All these research data will be very meaningful to uncover the remodeling process of endplates, to determine the resistance of endplates to the poor nutritional condition, and to elucidate the pattern of the reestablished endplate nutritional pathway after disc allografting. Collectively, this project is very meaningful to understand the degenerative mechanism of the disc allograft after transplantation which will be valuable for the development of new reparative approaches for more extensive application in clinics.

椎间盘同种异体移植已用于临床，并表现出较好的治疗效果，但移植后的椎间盘为何发生退变尚不明确。椎间盘主要经终板获得营养供给，但在终板营养通路建立之前，移植后的椎间盘一直处于无营养供给状态。营养缺失会导致细胞凋亡，进而诱发椎间盘退变。阐明椎间盘移植后终板变化将有可能解开椎间盘移植后的退变机理。在该项目中，我们首先将建立羊腰椎间盘同种异体移植模型，用动态增强MRI研究终板营养通路的重建模式；用UTE-MRI、Micro-CT、SEM-EDX mapping和组织学等研究骨终板和软骨终板的结构变化；最后我们将探讨冷冻保存椎间盘的软骨终板细胞对营养缺乏的耐受情况和以HIF-1α-HMGB1通路为主的凋亡机制。通过该研究，我们将从功能、结构和细胞水平上阐明椎间盘移植后终板营养通路重建模式、终板的结构和功能重构以及对营养缺乏的耐受情况。这些研究将逐步椎间盘移植后的退变机制，为研发新型修复技术奠定基础。

同种异体移植后的椎间盘发生退变的机制尚不明确。在终板通路建立之前，移植后的椎间盘一直处于无营养供给状态，营养缺失会诱发椎间盘退变。因此，阐明椎间盘移植后终板的结构和功能变化将有可能解开椎间盘移植后退变的机理。利用23只海南东山羊，我们成功建立了腰椎间盘同种异体移植模型，研究椎间盘移植后受体-移植物界面的骨愈合过程，探讨椎间盘移植后的再血管化机制，分析椎间盘移植后终板的结构变化，并与临床长期随访数据进行类比；此外，探讨了可用于促进椎间盘移植后骨-受体界面愈合的新材料，以加速终板通路的重建。我们发现，1）在移植1.5个月后，受体-移植物界面出现的新骨形成与骨愈合，经自然重构后，在移植12个月后受体-移植物界面的骨小梁结构与对正常骨小梁相比没有明显差异，受体-移植物界面的完整愈合，是椎间盘终板通路重建的前提。2）在移植1.5个月后，新生血管网络主要出现在骨终板区域和外纤维环；移植6个月后，在软骨终板和内纤维环出现新生血管；移植12个月后，血管网络逐渐向椎间盘中央靠拢，但始终没有侵袭到中央的软骨部分，骨终板区域始终是血管密度和面积最高的区域，这种再血管化模式与正常椎间盘的血管网有明显的差异。3）移植早期，终板的结构得到很好的维持，但移植6个月后，骨终板的皮质骨结构逐渐消失，最后基本被松质骨替代；同时，软骨终板经典的软骨陷窝出现大量的空腔化，软骨基质逐渐萎缩并出现不连续性，与骨终板的界面逐渐消失，这一发现和临床随访研究结果类似。4）我们初步探讨了软骨终板细胞的低氧环境下的生物学特性，还发现载锶和镁的骨脱细胞基质具有促进骨-受体界面愈合的潜能。通过本研究，我们阐明了椎间盘移植后受体-移植物界面的骨重构过程，这是维持椎间盘移植后长期稳定的基础；再血管化模式的改变和终板结构的消失可能是椎间盘移植后退变原因之一。该研究为椎间盘同种异体移植技术的临床推广和研发新型修复技术奠定基础。