Bertassoni Luiz E, Swain Michael V
a Division of Biomaterials and Biomechanics, Department of Restorative Dentistry, School of Dentistry , Oregon Health and Science University , Portland , OR , USA.
b Center for Regenerative Medicine , Oregon Health and Science University , Portland , OR , USA.
Connect Tissue Res. 2017 Sep;58(5):414-423. doi: 10.1080/03008207.2016.1235566. Epub 2016 Sep 22.
The structural organization of collagen from mineralized tissues, such as dentin and bone, has been a topic of debate in the recent literature. Recent reports have presented novel interpretations of the complexity of collagen type I at different hierarchical levels and in different tissues. Here, we investigate the nanostructural organization of demineralized dentin collagen following the digestion of non-collagenous components with a trypsin enzyme.
Dentin specimens were obtained from healthy third-molars, cut into small cubes, and polished down to 1 µm roughness. Samples were then demineralized with 10% citric acid for 2 min. Selected specimens were further treated with a solution containing 1 mg/ml trypsin for 48 hours at 37 °C (pH 7.9-9.0). Both untreated and trypsin digested samples were analyzed using SDS-PAGE, Field Emission Scanning Electron Microscopy (FE-SEM), and nanoindentation, where surface hardness and creep properties were compared before and after treatments.
FE-SEM images of demineralized dentin showed the banded morphology of D-periodical collagen type I, which upon enzymatic digestion with trypsin appeared to dissociate longitudinally, consistently unraveling ~20 nm structures (microfibril bundles). Such nanoscale structures, to the best of our knowledge, have not been characterized in dentin previously. Mechanical characterization via nanoindentation showed that the unraveling of such microfibril bundles affected the creep displacement and creep rate of demineralized dentin.
In summary, our results provide novel evidence of the organization of collagen type I from dentin, which may have important implications for the interaction of dental materials with the organic dentin matrix and the mechanical properties of mineralized tissues.
矿化组织（如牙本质和骨）中胶原蛋白的结构组织一直是近期文献中的一个争论话题。最近的报告对不同层次水平和不同组织中I型胶原蛋白的复杂性提出了新的解释。在此，我们研究了用胰蛋白酶消化非胶原蛋白成分后脱矿牙本质胶原蛋白的纳米结构组织。
从健康的第三磨牙获取牙本质标本，切成小方块，并抛光至粗糙度为1μm。然后将样品用10%柠檬酸脱矿2分钟。选定的标本在37°C（pH 7.9 - 9.0）下用含有1mg/ml胰蛋白酶的溶液进一步处理48小时。使用SDS - PAGE、场发射扫描电子显微镜（FE - SEM）和纳米压痕对未处理和经胰蛋白酶消化的样品进行分析，比较处理前后的表面硬度和蠕变特性。
脱矿牙本质的FE - SEM图像显示了I型D周期胶原蛋白的带状形态，在用胰蛋白酶进行酶消化后，其似乎纵向解离，持续解开约20nm的结构（微原纤维束）。据我们所知，这种纳米级结构以前在牙本质中尚未得到表征。通过纳米压痕进行的力学表征表明，这种微原纤维束的解开影响了脱矿牙本质的蠕变位移和蠕变率。
总之，我们的结果为牙本质中I型胶原蛋白的组织提供了新的证据，这可能对牙科材料与有机牙本质基质的相互作用以及矿化组织的力学性能具有重要意义。
