偏振控制二次谐波生成成像拉伸的胶原原纤维，原位揭示胶原变形通路
Polarization controlled second harmonic generation imaging of stretched collagen fibrils reveals collagen deformation pathway in situ
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中文总结 AI 辅助
本研究结合偏振控制二次谐波生成成像与原子力显微镜，原位揭示胶原原纤维内分子变形通路，发现三价交联可延迟胶原从产生SHG到不产生SHG的两态转变，且SHG分子应变大于D带应变
中文摘要 AI 辅助
作为承重组织基本组成单元的单一胶原原纤维的拉伸特性，已通过纳米力学技术和分子动力学模拟得到广泛研究。然而，原纤维内胶原分子的变形通路尚未被实验观测到，二价和三价酶促交联在调控该变形通路中发挥的作用也知之甚少。本研究采用偏振控制二次谐波生成（SHG）成像结合原子力显微镜（AFM），表征拉伸的单一胶原原纤维内胶原三螺旋的分子状态。从同一只动物的一对牛腿肌腱中提取原纤维，以比较富含未成熟二价交联的原纤维与富含成熟三价交联的原纤维。通过选取沿长度方向具有较大SHG强度梯度的原纤维，再用AFM对同一原纤维成像，我们能够将观测到的强度梯度与D带应变梯度及分子应变梯度关联起来，分子应变由SHG各向异性参数ρ估算。与此前肌腱尺度的研究结果相反，我们发现所有原纤维的SHG分子应变始终大于D带应变，且富含二价交联的原纤维中该差异最大。通过分析SHG发射体相对密度随分子应变的变化行为，我们观测到从产生SHG的状态到不产生SHG的状态存在两态转变，自由能垒介于6至10kBT之间，我们认为该转变对应胶原三螺旋超螺旋扭转的局部解旋，这可能发生在键断裂和SHG信号丧失之前。我们还表明，与二价交联相比，三价交联倾向于延迟该转变的起始。
英文摘要
The tensile properties of single collagen fibrils, the building block of load-bearing tissues, have been studied extensively by nanomechanical techniques and molecular dynamics simulation. However, the deformation pathway of collagen molecules within fibrils has not yet been observed experimentally. In addition, the role played by divalent and trivalent enzymatic crosslinks in modulating this deformation pathway is poorly understood. Here we used polarization controlled second harmonic generation (SHG) imaging combined with atomic force microscopy (AFM) to characterize the molecular state of collagen triple helices within stretched single collagen fibrils. The fibrils were extracted from a pair of bovine leg tendons from the same animal in order to compare fibrils with a high amount of immature divalent crosslinks to fibrils with a high amount of mature trivalent crosslinks. By selecting fibrils with a large SHG intensity gradient along their length and then imaging the same fibrils by AFM we were able to link the observed intensity gradient with a gradient in D-band strain and a gradient in molecular strain as estimated from the SHG anisotropy parameter rho. In contrast to previous studies at the tendon scale, we observed that the SHG molecular strain is always larger than the D-band strain for all fibrils with this difference being largest for fibrils rich in divalent crosslinks. By analyzing the behavior of the relative density of SHG emitters as a function of molecular strain, we observe a two-state transition from an SHG producing to a non SHG producing state with a free energy barrier between 6 and 10 kBT that we propose corresponds to the local untwisting of the collagen triple helix superhelical twist which likely preceded bond rupture and loss of the SHG signal. We also show that trivalent crosslinks tend to delay the transition onset compared to divalent crosslinks.
