title: Comparative study of demineralized collagen degradation determined by hydroxyproline assay and microscopic depth measurement.
abstract: BACKGROUND
  Quantification of collagen degradation is an important parameter to evaluate dentin caries progression or the efficacy of caries prevention aid. The aim of this study was to validate the simple light microscopic technique (LM) to evaluate collagen degradation by comparing with hydroxyproline assay technique (HPN).
  
  
  METHODS
  Bovine root dentin blocks were embedded in acrylic resin, polished and covered with nail varnish except a 1.5 × 2.5mm window. The specimens were demineralized in acetate buffer (pH 4.3) for 3 days to create incipient lesions and were exposed to collagenase enzyme for 6, 9 and 16 h. The specimens were sectioned into thin sections (200-220 μm) to measure the degraded depth of collagen matrix by LM. The enzyme solutions were allocated to HPN assay using the simplified chloramines-T method. Correlation between LM and HPN was evaluated by Pearson correlation analysis. Anti-collagen degradation efficacy of 0.12% chlorhexidine (CHX) was evaluated by LM.
  
  
  RESULTS
  The depths of the degraded collagen and amount of hydroxyproline in 3 exposure periods were 27.8 ± 3.8 μm and 28.7 ± 4.2 μg for 6h, 48.1 ± 8.6 μm and 45.3 ± 6.1 μg for 9h, and 74.2 ± 9.7 μm and 71.3 ± 8.0 μg for 16 h, respectively. A significantly positive correlation (r=0.94, CI: 0.88-0.97, p<0.0001) was observed between LM and HPN and incubation time showed a linear correlation with amount of collagen degradation (R(2)=0.92). The CHX group (28.6 ± 3.3 μm) showed significantly lower collagen degradation than that of control group (53.1 ± 7.8 μm: p<0.01).
  
  
  CONCLUSIONS
  The LM might be a reliable and simplified method to evaluate collagen degradation.
authors.affiliation: Cariology and Operative Dentistry, Department of Restorative Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; International Exchange Center, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Electronic address: soheltmd@gmail.com.
authors.forename: Md Sofiqul
authors.initials: MS
authors.lastname: Islam
authors.affiliation: Cariology and Operative Dentistry, Department of Restorative Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Sahar Jameel
authors.initials: SJ
authors.lastname: Khunkar
authors.affiliation: Cariology and Operative Dentistry, Department of Restorative Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Syozi
authors.initials: S
authors.lastname: Nakashima
authors.affiliation: Department of Restorative Dentistry, School of Dentistry, University of Washington, 1959 NE Pacific St B162, Seattle, WA 98195, USA.
authors.forename: Alireza
authors.initials: A
authors.lastname: Sadr
authors.affiliation: Cariology and Operative Dentistry, Department of Restorative Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Nikaido
authors.affiliation: Cariology and Operative Dentistry, Department of Restorative Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Tagami
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002710:Chlorhexidine / Q000008:administration & dosage / Q000494:pharmacology; D003094:Collagen / Q000032:analysis / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism; D003731:Dental Caries / Q000000981:diagnostic imaging / Q000378:metabolism*; D003804:Dentin / Q000737:chemistry / Q000378:metabolism; D006909:Hydroxyproline / Q000032:analysis / Q000378:metabolism*; D008859:Microscopy, Polarization / Q000379:methods; D017001:Tooth Demineralization / Q000000981:diagnostic imaging* / Q000201:enzymology / Q000378:metabolism; D014092:Tooth Root / Q000737:chemistry
chemical_list: D000180:Acrylic Resins; D003094:Collagen; D017364:Collagenases; D002710:Chlorhexidine; D006909:Hydroxyproline
keywords: Collagen-degradation; Demineralization; Dentin; Hydroxyproline; Microscopy