Collagen Degradation by Host-derived Enzymes during Aging
D.H. Pashley1,*,
F.R. Tay2,
C. Yiu2,
M. Hashimoto3,
L. Breschi4,
R.M. Carvalho5, and
S. Ito6
1 Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, GA 30912, USA;
2 Paediatric Dentistry and Orthodontics, University of Hong Kong, Hong Kong, SAR, China;
3 Division of Pediatric Dentistry, Hokkaido University, Graduate School of Dental Medicine, Kita 13 Nishi 7, Kita-ku, Sapporo 060-8586, Japan;
4 Department of Surgical Sciences, University of Trieste, Italy;
5 Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry, University of São Paulo, Brazil; and
6 Department of Operative Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu, 061-0293, Japan;
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Figure 1. Transmission electron micrographs of control acid-etched dentin. (A) Unstained, undemineralized TEM micrograph of phosphoric-acid-etched dentin after being aged in artificial saliva for 24 hrs. A 5- to 6-µm-thick layer (between open arrows) of demineralized collagen matrix (CM) can be observed. D, undemineralized dentin; E, epoxy resin. (B) Higher magnification of the completely demineralized surface region of Fig. 1A , but which was stained for collagen. Unraveling of the cut collagen fibrils (pointers) resulted in the appearance of microfibrillar strands (i.e., partially denatured collagen) along the surface of the cut dentin. Cross-banding can be identified from the underlying intact collagen fibrils. (C) Stained demineralized TEM micrograph of the surface of phosphoric-acid-etched dentin after being aged in artificial saliva for 90 days. The thickness of the collagen matrix was first identified from unstained, undemineralized sections (not shown) and was found to be similar to that observed in the 24-hour specimens. The unraveled microfibrils disappeared from the surface of the cut dentin, and the exposed ends of the collagen fibrils appeared blunted (pointers).
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Figure 2. Transmission electron micrographs of acid-etched dentin incubated in artificial saliva for 250 days. (A) Unstained, undemineralized micrograph of phosphoric-acid-etched dentin that was aged in artificial saliva for 250 days. The entire thickness of collagen matrix completely disappeared, exposing the highly irregular demineralization front of the original acid-etched dentin. D, undemineralized dentin; E, epoxy resin. (B) Another unstained, undemineralized micrograph from the 250-day artificial saliva group showing the presence of a thin remnant layer of unstained collagen fibrils (pointer) above the demineralized matrix. D, undemineralized dentin; E, epoxy resin. (C) Stained, demineralized micrograph taken from the same group, showing the disappearance of the bulk of the demineralized collagen matrix. A 1-µm-thick remnant zone of sparsely arranged, banded collagen fibrils (pointer) can be observed over the previously unetched, laboratory-demineralized dentin (D). E, epoxy resin; T, dentinal tubule.
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Figure 3. Transmission electron micrographs of acid-etched dentin incubated in oil for 250 days. (A) Unstained, undemineralized micrograph of phosphoric-acid-etched dentin that was aged in mineral oil for 250 days. The demineralized collagen matrix (CM) was retained, but was collapsed and appeared thinner due to dehydration in the mineral oil. D, undemineralized dentin. Slightly electron-dense patches that appeared on the epoxy resin (E) were water marks that represented minute water droplets that evaporated from the surface of the hydrophobic epoxy resin following retrieval of the carbon- and formvar-coated copper grid from the water reservoir of the diamond knife. (B) Higher magnification of the matrix in CM in Fig. 3A, after double-staining with phosphotungstic acid (PTA) and uranyl acetate to reveal structural detail of collagen fibrils. Intact intertubular collagen fibrils observed 0.5 µm beneath the cut dentin surface revealed a three-dimensional arrangement with intact cross-banding.
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Figure 4. Transmission electron micrographs of acid-etched dentin incubated in protease inhibitors for 250 days. (A) Unstained, undemineralized micrograph of phosphoric-acid-etched dentin that was aged in artificial saliva containing protease inhibitors for 250 days. Unlike specimens that were aged for the same period in artificial saliva without protease inhibitors, in which the bulk of the demineralized collagen matrix was dissolved, the demineralized collagen matrix (CM) in the proteolytic enzyme inhibitor group was preserved, and there were crystallites (pointers) deposited along the cut surface of the dentin as well as along the demineralization front (pointers). The internal surfaces (demarcated by arrowheads) of the dentinal tubules (T) were devoid of these crystallites. D, undemineralized dentin; E, epoxy resin. (B) Higher magnification of completely demineralized sections stained for collagen, taken from the surface of 250-day protease-inhibited, aged, acid-etched dentin. Similar to specimens that were aged for 24 hrs (control) in artificial saliva, microfibrillar strands (pointers) that were derived from the unraveling of the collagen fibrils were preserved. The acidity of the PTA stain dissolved the surface crystals.
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