Removal of dentin non-collagenous structures results in the unraveling of microfibril bundles in collagen type I

ABSTRACT

Aims: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

KEYWORDS:

• Collagen
• dentin
• microfibril
• proteoglycans
• trypsin

Acknowledgments

We thank Dr. Catherine Rathsam and Matheus Kury for assistance with 565 SDS-PAGE analyses and Prof. Christopher Little for valuable discussions of proteoglycans in load bearing tissues.

Funding

This research was supported by the Australian Research Council (ARC) (DP120104837), Australian Dental Research Foundation (ADRF), grant no. 46/2009 and 36/2010, the Medical Research Foundation of Oregon (MRFO), and the National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR; R01DE26270).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Additional information

Funding

This research was supported by the Australian Research Council (ARC) (DP120104837), Australian Dental Research Foundation (ADRF), grant no. 46/2009 and 36/2010, the Medical Research Foundation of Oregon (MRFO), and the National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR; R01DE26270).