Introduction

Structural changes in the collagen II architecture of osteoarthritis (OA) are poorly understood, which is a large shortcoming in the early diagnosis of this disease. Though degradation can be simulated by enzymes including trypsin and bacterial collagenase, the specific structural features of each digestion and their relationship to naturally occurring OA remain unclear.

Experimental design

We used collagen sensitive/specific Second Harmonic Generation (SHG) microscopy in conjunction with optical scattering measurements to probe the resulting architecture changes in bovine knee cartilage upon trypsin and collagenase degradation. Image features extracted from SHG images were used to train a linear discriminant (LD) model capable of classifying enzymatic degradation, which was then applied to human cartilage with varied modified Mankin histological scores.

Results

The treatment of cartilage with these enzymes resulted in more disorganized collagen structure, where this effect was greatest with collagenase treatment. Using the LD model, we classified the control and degraded tissues in the three zones with >92% accuracy, showing that these enzymes have distinct activity on the collagen assembly. Application of the LD model to human cartilage indicated that collagenase effects were more representative of in vivo degeneration and were also consistent with damage beginning at the articular surface and progressing into deeper zones.

Conclusions

SHG and optical scattering measurements successfully delineate trypsin and collagenase degradation and suggest that collagen alterations in human OA are better simulated by the latter mechanism. These results lay the groundwork for using high-resolution SHG and optical scattering as an earlier diagnostic tool than is currently available.

中文翻译：

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通过二次谐波显微镜评估骨关节炎酶降解模型中胶原蛋白的变化

介绍

骨关节炎 (OA) 的胶原 II 结构的结构变化知之甚少，这是该疾病早期诊断的一大缺点。虽然降解可以通过包括胰蛋白酶和细菌胶原酶在内的酶来模拟，但每种消化的具体结构特征及其与天然 OA 的关系仍不清楚。

实验设计

我们使用胶原敏感/特异性二次谐波 (SHG) 显微镜结合光学散射测量来探测胰蛋白酶和胶原酶降解后牛膝关节软骨的结构变化。从 SHG 图像中提取的图像特征用于训练能够对酶降解进行分类的线性判别 (LD) 模型，然后将其应用于具有不同改良 Mankin 组织学评分的人类软骨。

结果

用这些酶处理软骨导致胶原蛋白结构更加混乱，其中这种效果在胶原酶处理时最大。使用 LD 模型，我们以 >92% 的准确度对三个区域中的对照和退化组织进行分类，表明这些酶对胶原蛋白组装具有不同的活性。将 LD 模型应用于人类软骨表明胶原酶效应更能代表体内退化，并且也与从关节表面开始并进展到更深区域的损伤一致。

结论

SHG 和光学散射测量成功地描绘了胰蛋白酶和胶原酶的降解，并表明后一种机制可以更好地模拟人类 OA 中的胶原改变。这些结果为使用高分辨率 SHG 和光学散射作为比目前可用的更早的诊断工具奠定了基础。