A-Traumatic Tooth Extraction Based on an Optimized Collagenase

Objectives: Collagenases main function is the breaking of the triple helix collagen. Range of applications can benefit from optimized collagenase proteins having higher activity and stability. The aim of the present study is to engineer an optimized collagenase variant that can more efficiently break down the periodontal ligament collagen, enabling the extraction of teeth without the need to apply exaggerated mechanical force as it is done today.
Methods: Collagenase G of Clostridium histolyticum was expressed in E.coli and purified. Activity assay to monitor the degradation of collagen was developed enabling the evaluation of collagenase activity in a 96 wells format to support screening of optimized protein variants from a genetic library. The assay is highly selective, enabling the direct detection of collagenase activity directly on collagen.
Ex-situ activity was tested on tooth extraction model of Porcine mandible, by measuring the vertical force, using Instron universal testing system model 4500 (Instron).
Genetic library was constructed based on Collagenase G gene via the application of GeneMorph II Random Mutagenesis Kit (Agilent), adjusted to produce an average of 6 non-synonymous mutations per gene.
Results: In this study we established the activity of collagenase G on periodontal ligament collagen. Our data show reduction in the required force for the extraction of tooth in a model of Porcine mandible. In addition, we screened and discovered preliminary collagenase variants with improved activity. Clone sequencing revealed a unique Phe782 to Ser mutation.
Conclusions: Degradation of the triple helix collagen is important in a range of biotechnology and medical applications. Our results show a promising assay to screen for optimized collagenase and its application towards a-traumatic tooth extraction.