Department of Orthopaedic Surgery, Academic Medical Center, University of Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands; Department of Orthopedic Surgery, VU University Medical Center, Amsterdam Movement Sciences, The Netherlands.
Materials and Engineering Research Institute, Sheffield Hallam University, Sheffield, UK.
Osteoarthritis Cartilage. 2018 Oct;26(10):1400-1408. doi: 10.1016/j.joca.2018.06.003. Epub 2018 Jun 20.
Mechanical overloading induces a degenerative cell response in the intervertebral disc. However, early changes in the extracellular matrix (ECM) are challenging to assess with conventional techniques. Fourier Transform Infrared (FTIR) imaging allows visualization and quantification of the ECM. We aim to identify markers for disc degeneration and apply these to investigate early degenerative changes due to overloading and katabolic cell activity.
Three experiments were conducted; Exp 1.: In vivo, lumbar spines of seven goats were operated: one disc was injected with chondroitinase ABC [cABC (mild degeneration)] and compared to the adjacent disc (control) after 24 weeks. Exp 2a: Ex vivo, caprine discs received physiological loading (n = 10) or overloading (n = 10) in a bioreactor. Exp 2b: Cell activity was diminished prior to testing by freeze-thaw cycles, 18 discs were then tested as in Exp 2a. In all experiments, FTIR images (spectral region: 1000-1300 cm) of mid-sagittal slices were analyzed using multivariate curve resolution.
In vivo, FTIR was more sensitive than biochemical and histological analysis in identifying reduced proteoglycan content (P = 0.046) and increased collagen content in degenerated discs (P < 0.01). Notably, FTIR analysis additionally showed disorganization of the ECM, indicated by increased collagen entropy (P = 0.011). Ex vivo, the proteoglycan/collagen ratio decreased due to overloading (P = 0.047) and collagen entropy increased (P = 0.047). Cell activity affected collagen content only (P = 0.044).
FTIR imaging allows a more detailed investigation of early disc degeneration than traditional measures. Changes due to mild overloading could be assessed and quantified. Matrix remodeling is the first detectable step towards intervertebral disc degeneration.
机械性超负荷会导致椎间盘发生退行性细胞反应。然而，传统技术难以评估细胞外基质（ECM）的早期变化。傅里叶变换红外（FTIR）成像可实现 ECM 的可视化和定量分析。我们旨在寻找椎间盘退变的标志物，并应用这些标志物来研究因超负荷和分解代谢细胞活性导致的早期退行性变化。
进行了三项实验；实验 1：在体内，对七只山羊的腰椎进行手术：一只椎间盘注射软骨素酶 ABC[cABC（轻度退变）]，24 周后与相邻椎间盘（对照）进行比较。实验 2a：在体外，羊椎间盘在生物反应器中接受生理负荷（n=10）或超负荷（n=10）。实验 2b：在测试前通过冻融循环降低细胞活性，然后将 18 个椎间盘按照实验 2a 进行测试。在所有实验中，使用多元曲线分辨法对中矢状切片的 FTIR 图像（光谱区域：1000-1300cm）进行分析。
在体内，FTIR 比生化和组织学分析更能敏感地识别退变椎间盘中蛋白聚糖含量降低（P=0.046）和胶原含量增加（P<0.01）。值得注意的是，FTIR 分析还显示 ECM 的紊乱，表现为胶原熵增加（P=0.011）。在体外，由于超负荷，蛋白聚糖/胶原比值降低（P=0.047），胶原熵增加（P=0.047）。细胞活性仅影响胶原含量（P=0.044）。
FTIR 成像比传统方法更能详细地研究早期椎间盘退变。可以评估和量化轻度超负荷引起的变化。基质重塑是椎间盘退变的第一个可检测步骤。
