The role of extracellular matrix in collagen biomineralization

Lausch, Alexander (Institute for Biomaterials and Biomedical Engineering)
Sone, Eli (Institute for Biomaterials and Biomedical Engineering)

Introduction

Collagen biomineralization is a complex process and the controlling factors, at the molecular level, are not well understood. A particularly elegant example of biological control over collagen mineralization is found in the periodontium, the set of tissues involved in tooth anchorage. The periodontium is composed of both mineralized and unmineralized tissues (in close proximity) and as such, is an excellent environment for investigation into the controlling mechanisms of mineralization. More specifically, the periodontal ligament (PDL) becomes mineralized along a sharp front of about 200nm at the cementum/PDL junction. It is thought that this fine spatial control over mineralization must be due to extracellular matrix (ECM) components, specifically, highly acidic/anionic non-collagenous proteins (NCPs). However, the role of these proteins in collagen biomineralization is not known. Here, employ a recently developed model of collagen biomineralization [1], using mouse periodontium, to display ECM control over mineralization. Remineralized periodontium retains spatial control over mineralization, and thus we are able to probe the controlling factors. Using protein extraction methods we can show how specific ECM components are involved in directing physiological mineralization at the PDL/cementum junction. Understanding the mechanisms involved in selective mineralization is necessary toward the development of bioactive scaffolds for bone implants, or for the treatment of periodontitis and other mineralization diseases.

Materials and Methods

Male CD1 mice are sacrificed by cervical dislocation in accordance with University of Toronto animal use protocol 20010383. Mandibles are removed and immersed in a solution of glutaraldehyde, paraformaldehyde, and EDTA to simultaneously fix and demineralize the tissue. Ultra-thin sections of demineralized mandible are sectioned under cryo-conditions to show bone, ligament and dentin. Sections are exposed to solutions, which are supersaturated with respect to hydroxyapatite and stabilized with polyaspartic acid, at 37°C.

Results

Previously, we have shown that simultaneously fixed and demineralized tissues are capable of directing remineralization with near-native selectivity. To elucidate the mechanisms controlling mineralization we use GuHCl extraction, and different enzymatic digestions to remove specific ECM components, and observe their effects on remineralization. We tested the effect of the removal of all non-collagenous proteins (trypsin/GuHCl); the removal of all proteoglycans (chondroitinase, hyaluronidase); and complete dephosphorylation (alkaline phosphatase). Using amino acid analysis we have shown that trypsin is capable of removing 95% of NCPs from periodontal tissues. The removal of all NCPs, by trypsin digestion, or GuHCl treatment, results in a dramatically reduced rate of remineralization in dentin and cementum. Moreover, cementum is particularly sensitive to this treatment and resembles periodontal ligament. Further, we have shown that remineralization is slightly retarded in all naturally mineralized tissues after dephosphorylation. We are currently working on quantifying the dephosphorylation by immunohistochemistry. The removal of glycosaminoglycans by enzymatic digestion has been shown to be at least 50% efficient, as shown by a DMMB assay [2]. Despite the removal of the majority of these macromolecules, there is little to no effect on remineralization.

Discussion and Conclusion

The use of our previously developed model of collagen mineralization allows us to manipulate the extracellular matrix to better understand the components involved in biomimetic remineralization of the tooth root interface. Using enzymatic digestions and protein extractions, we have shown that the ECM is directly involved in controlling remineralization of the periodontium, and that its role seems to be the promotion of mineral deposition in naturally mineralizing tissues. No treatments seem to have any effect on PDL, indicating that mineral inhibition, in our model, is not as important as promotion. The removal of glycosaminoglycans, molecules thought to inhibit mineralization, seems to have no effect on remineralization. It is possible that the efficacy of the digests can have an effect on the remineralization and that a removal of >50% is not significant enough to show a significant effect on remineralization. However, our quantification technique underestimates the amount of removal as it only assays sulfated GAGs. We are currently working to improve the efficacy of this digestion. Understanding biological control over collagen mineralization can aide in the engineering of hard/soft tissue interfaces. Periodontitis is an inflammatory disease, which is characterized by the degradation of the ligament/cementum junction, due to bacterial invasion, and is the leading cause of tooth loss worldwide. Current attempts at periodontal regeneration have been unsuccessful at predictably regenerating tooth attachment. Bioactive scaffolds have the potential for the directed regeneration of this interface and subsequent tooth reattachment.

Acknowledgements

This research is supported by NSERC, the CIHR Institute of Musculoskeletal Health and Arthritis, and a Faculty of Dentistry Junior Faculty Research Advancement Award to EDS. AJL is grateful to the NSERC CREATE Program in Regenerative Medicine for a doctoral fellowship.

References

[1] Lausch, A.J., Quan, B.D., Miklas, J.W., Sone, E.D. Extracellular matrix control of collagen mineralization in vitro (2013) Advanced Functional Materials, 23 (39), pp. 4906-4912. [2] Farndale, R.W., Buttle, D.J., Barrett, A.J. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue (1986) BBA - General Subjects, 883 (2), pp. 173-177.

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