Type IV Collagen is a Novel DEJ Biomarker that is Reduced by Radiotherapy

Abstract

The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentinenamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy -teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5-to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its wellknown fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ instability observed following oral cancer radiotherapy.

Structural Analysis of Enamel in Teeth from Head-and-Neck Cancer Patients Who Underwent Radiotherapy

“…In this regard, we hypothesize that ionizing radiation could cause nonselective oxidation of organic components including decarboxylation and finally proteolysis of noncollagenous enamel proteins that were previously present in the interprismatic spaces. This proteolytic mechanism could perhaps occur in a similar way as radiotherapy breaks the links that maintain the ultrastructural organization of organic components in other human tissues [Mitchell and Logan, 1998;Hübner et al, 2005;McGuire et al, 2014a]. On the other hand, we can also hypothesize that such a proteolytic effect could be produced by indirect enzyme activation during or after radiation therapy.…”

“…A series of in vitro studies in this context have shown that high doses of ionizing radiation could impair the stability of enamel, dentin, the dentino-enamel junction, human dental pulp, and the periodontal ligament [Pioch et al, 1992;Kielbassa et al, 2006;Konstantonis et al, 2013;McGuire et al, 2014a].…”

Radiotherapy Reduces Microhardness and Mineral and Organic Composition, and Changes the Morphology of Primary Teeth: An in vitro Study

“…With respect to collagen, the authors showed the importance of type IV collagen in the dentin-enamel junction. Reduction of this component may represent biochemical changes and instability of the dentin-enamel junction of irradiated teeth [McGuire et al, 2014].…”

Radiotherapy Activates and Protease Inhibitors Inactivate Matrix Metalloproteinases in the Dentinoenamel Junction of Permanent Teeth

“…In contrast, type IV collagen loss offers a possible biochemical explanation for the instability observed postradiotherapy in the DEJ. Another region assessed was the coronary dentin, which contains type I collagen and is well-known for the activity of MMP-2 and MMP-9 in processes such as dental erosion and caries [McGuire et al, 2014a].…”

“…Currently, there are few studies in the literature that have evaluated MMP activity in teeth postradiotherapy [McGuire et al, 2014b;Gomes-Silva et al, 2017a, b] and one study that found reduced amounts of collagen type IV [McGuire et al, 2014a]. With regard to the inactivation of MMPs by treatment with protease inhibitors, including green tea or polyphenol epigallocatechin-3-gallate (EGCG) [Kato et al, 2010a[Kato et al, , 2012Buzalaf et al, 2014], chlorhexidine (CHX) [Kato et al, 2010a[Kato et al, , 2012, and sodium fluoride (NaF) , no studies in the literature have been conducted with postradiotherapy teeth.…”