Dhiman Manmeet S, Bader Taylor J, Ponjevic Dragana, Salo Paul T, Hart David A, Swamy Ganesh, Matyas John R, Duncan Neil A
Department of Biomedical Engineering University of Calgary Calgary Alberta Canada.
McCaig Institute for Bone and Joint Health University of Calgary Calgary Alberta Canada.
JOR Spine. 2024 Jul 31;7(3):e1359. doi: 10.1002/jsp2.1359. eCollection 2024 Sep.
Degenerative disc disease (DDD) is accompanied by structural changes in the intervertebral discs (IVD). Extra-cellular matrix degradation of the annulus fibrosus (AF) has been linked with degeneration of the IVD. Collagen is a vital component of the IVD. Collagen hybridizing peptide (CHP) is an engineered protein that binds to degraded collagen, which we used to quantify collagen damage in AF. This method was used to compare AF samples obtained from donors with no DDD to AF samples from patients undergoing surgery for symptomatic DDD.
Fresh AF tissue was embedded in an optimal cutting temperature compound and cryosectioned at a thickness of 8 μm. Hematoxylin and Eosin staining was performed on sections for general histomorphological assessment. Serial sections were stained with Cy3-conjugated CHP and the mean fluorescence intensity and areal fraction of Cy3-positive staining were averaged for three regions of interest (ROI) on each CHP-stained section.
Increases in mean fluorescence intensity ( = 0.0004) and percentage of positively stained area ( = 0.00008) with CHP were detected in DDD samples compared to the non-DDD samples. Significant correlations were observed between mean fluorescence intensity and percentage of positively stained area for both non-DDD ( = 0.98, = 5E-8) and DDD ( = 0.79, = 0.0012) samples. No significant differences were detected between sex and the lumbar disc level subgroups of the non-DDD and DDD groups. Only tissue pathology (non-DDD versus DDD) influenced the measured parameters. No three-way interactions between tissue pathology, sex, and lumbar disc level were observed.
These findings suggest that AF collagen degradation is greater in DDD samples compared to non-DDD samples, as evidenced by the increased CHP staining. Strong positive correlations between the two measured parameters suggest that when collagen degradation occurs, it is detected by this technique and is widespread throughout the tissue. This study provides new insights into the structural alterations associated with collagen degradation in the AF that occur during DDD.
椎间盘退变（DDD）伴有椎间盘（IVD）的结构变化。纤维环（AF）的细胞外基质降解与IVD退变有关。胶原蛋白是IVD的重要组成部分。胶原杂交肽（CHP）是一种工程蛋白，可与降解的胶原蛋白结合，我们用它来量化AF中的胶原损伤。该方法用于比较从无DDD的供体获得的AF样本与因症状性DDD接受手术的患者的AF样本。
将新鲜的AF组织包埋于最佳切片温度化合物中，切成8μm厚的冰冻切片。对切片进行苏木精和伊红染色以进行一般组织形态学评估。连续切片用Cy3标记的CHP染色，对每个CHP染色切片上的三个感兴趣区域（ROI）的Cy3阳性染色的平均荧光强度和面积分数进行平均。
与非DDD样本相比，DDD样本中CHP的平均荧光强度（=0.0004）和阳性染色面积百分比（=0.00008）增加。在非DDD（=0.98，=5E-8）和DDD（=0.79，=0.0012）样本中，平均荧光强度与阳性染色面积百分比之间均观察到显著相关性。非DDD组和DDD组的性别和腰椎间盘水平亚组之间未检测到显著差异。只有组织病理学（非DDD与DDD）影响测量参数。未观察到组织病理学、性别和腰椎间盘水平之间的三因素相互作用。
这些发现表明，与非DDD样本相比，DDD样本中AF胶原降解更严重，CHP染色增加证明了这一点。两个测量参数之间的强正相关表明，当胶原降解发生时，该技术可检测到，且其在整个组织中广泛存在。本研究为DDD期间AF中与胶原降解相关的结构改变提供了新的见解。
