title: Removal of dentin non-collagenous structures results in the unraveling of microfibril bundles in collagen type I.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.|c Department of Biomedical Engineering , School of Medicine, Oregon Health and Science University , Portland , OR , USA.|d Bioengineering Laboratory, Faculty of Dentistry , University of Sydney , Sydney , NSW , Australia.
authors.forename: Luiz E
authors.initials: LE
authors.lastname: Bertassoni
authors.affiliation: d Bioengineering Laboratory, Faculty of Dentistry , University of Sydney , Sydney , NSW , Australia.|e Bioclinical Sciences Department , Faculty of Dentistry, University of Kuwait , Kuwait.
authors.forename: Michael V
authors.initials: MV
authors.lastname: Swain
mesh_terms: D024042:Collagen Type I / Q000737:chemistry*; D003804:Dentin / Q000737:chemistry*; D005109:Extracellular Matrix / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D008963:Molar / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D024042:Collagen Type I
keywords: Collagen; dentin; microfibril; proteoglycans; trypsin