IADR Abstract Archives
Isolation of Enamel Matrix Proteins From MMP-20 Deficient Murine Molars
Objectives: Radiotherapy for oral cancer has many post-treatment complications including delamination of enamel from dentin at the dentin-enamel junction (DEJ). Near the DEJ an enamel organic matrix layer which may contribute to DEJ stability has been identified. Previously we showed that MMP-20 is present in exposed teeth and is resistant to radiotherapy. We have hypothesized that MMP-20 may degrade enamel matrix proteins leading to destabilization of the enamel-dentin bond and subsequent delamination of enamel from dentin. Because there is no way to directly test MMP-20 function in humans, genetically engineered mouse models were investigated for their potential. A protocol was developed for extracting enamel proteins from mature mouse molars. These were then compared to enamel proteins of human molars by western blotting.
Methods: A protocol was developed to isolate enamel proteins from molars derived from individual wild type, MMP-20 heterozygous, and MMP-20 null mice. Western blotting was used to determine expression and molecular sizes of MMP-20, type VII collagen, type IV collagen, and laminin.
Results: Compared to wild type molars, amount of MMP-20 was halved in MMP-20 heterozygous and absent in MMP-20 null molars (Figure), validating the protocol. Type VII collagen, type IV collagen, and laminin were detected in all molars but an additional protein extraction step was needed to efficiently extract high molecular forms.
Conclusions: Enamel matrix proteins can be successfully extracted from mouse molars and analyzed. Two extraction steps were necessary to efficiently recover high molecular weight forms and less than one microgram of total protein was needed for detection. Enamel protein composition in wild type and MMP-20 heterozygous mice is similar to that of humans. Therefore, some transgenically modified mouse models may have good potential for answering clinical questions related to the role of enamel proteins in delamination following radiotherapy. (Funded by UMKC SOD Summer Scholars Program)
Methods: A protocol was developed to isolate enamel proteins from molars derived from individual wild type, MMP-20 heterozygous, and MMP-20 null mice. Western blotting was used to determine expression and molecular sizes of MMP-20, type VII collagen, type IV collagen, and laminin.
Results: Compared to wild type molars, amount of MMP-20 was halved in MMP-20 heterozygous and absent in MMP-20 null molars (Figure), validating the protocol. Type VII collagen, type IV collagen, and laminin were detected in all molars but an additional protein extraction step was needed to efficiently extract high molecular forms.
Conclusions: Enamel matrix proteins can be successfully extracted from mouse molars and analyzed. Two extraction steps were necessary to efficiently recover high molecular weight forms and less than one microgram of total protein was needed for detection. Enamel protein composition in wild type and MMP-20 heterozygous mice is similar to that of humans. Therefore, some transgenically modified mouse models may have good potential for answering clinical questions related to the role of enamel proteins in delamination following radiotherapy. (Funded by UMKC SOD Summer Scholars Program)
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