Oral cancer radiotherapy affects enamel microhardness and associated indentation pattern morphology.

OBJECTIVES: The aim of this study is to determine the effects of in vitro and in vivo high-dose radiotherapy on microhardness and associated indentation pattern morphology of enamel. MATERIALS AND METHODS: The inner, middle, and outer microhardness of enamel was evaluated using three experimental groups: control (non-radiated); in vitro irradiated; in vivo irradiated. In vitro specimens were exposed to simulated radiotherapy, and in vivo specimens were extracted teeth from oral cancer patients previously treated with radiotherapy. Indentations were measured via SEM images to calculate microhardness values and to assess the mechanomorphological properties of enamel before and after radiotherapy. RESULTS: Middle and outer regions of enamel demonstrated a significant decrease in microhardness after in vitro and in vivo irradiation compared to the control group (p < 0.05). Two indentation patterns were observed: pattern A-presence of microcracks around indent periphery, which represents local dissipation of deformation energy; pattern B-clean, sharp indents. The percentage of clean microindentation patterns, compared to controls, was significantly higher following in vitro and in vivo irradiation in all enamel regions. The highest percentage of clean microindentations (65%) was observed in the in vivo irradiated group in the inner region of enamel near the dentin-enamel junction. CONCLUSIONS: For the first time, this study shows that in vitro and in vivo irradiation alters enamel microhardness. Likewise, the indentation pattern differences suggest that enamel may become more brittle following in vitro and in vivo irradiation. CLINICAL RELEVANCE: The mechanomorphological property changes of enamel following radiation may be a contributory component of pathologic enamel delamination following oral cancer radiotherapy.

Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

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 dentin-enamel 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 well-known 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.

Extracts of irradiated mature human tooth crowns contain MMP-20 protein and activity.

OBJECTIVES: We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. MATERIALS AND METHODS: Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0-6 months at 37°C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. RESULTS: Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentine and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr=43, 41, 24 and 22kDa and was immunolocalized predominantly to the morphological dentine enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70Gy, subsequent incubation at 37°C for 3-6 months with or without prior irradiation caused the proportion of Mr=24-22kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70Gy radiation also contained relatively more 24 and 22kDa MMP-20 than those of healthy age-related teeth. CONCLUSION: MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentine-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy.