Collagen degradation and preservation of MMP-8 activity in human dentine matrix after demineralization.
OBJECTIVE
Dental caries is a process driven by acids produced by oral microorganisms followed by degradation of the dentine collagen matrix by proteolytic enzymes. Matrix metalloproteinases (MMPs) have been suggested to contribute to caries by degrading collagen. The aim of this study was to develop a method for generating demineralized dentine matrix substrate (DDM) maintaining MMP-8 bioactivity and no interference with later assays. Such a substrate would allow study of the effects of various treatments on MMP-8 activity and collagen degradation in demineralized dentine.


METHODS
Human dentine was powderized in a tissue grinder and frozen (-80°C). The powder was demineralized in dialysis tubes, using EDTA or acetic acid. The demineralized dentine matrix (DDM) was harvested and analyzed for collagen content using SDS-PAGE. The DDM was subsequently suspended in PBS or TESCA buffer. Protein, MMP-8 (ELISA) and collagen (HYP) was analyzed directly or after 1 wk.


RESULTS
EDTA or acid demineralization of dentine using dialysis yielded a substrate rich in collagen coupled with preserved MMP-8 activity. Collagen degraded in room temperature, assessed by higher HYP amounts in the soluble fraction of DDM after one wk, indicating that the methods used preserved active DDM-components after the demineralization process.


CONCLUSIONS
The presented demineralization methods both provided insoluble DDM substrates suitable for further intervention studies. However, it was found that the substrates differed depending on the demineralization method and buffers used. This needs further study to find an optimal technique for generating DDM with retained proteins as well as enzymatic bioactivity.
Department of Cariology, Faculty of Odontology, Malmö University, Sweden. Electronic address: anders.hedenbjork.lager@mah.se.
Anders
A
Hedenbjörk-Lager
Department of Cariology, Faculty of Odontology, Malmö University, Sweden.
Kristina
K
Hamberg
Research Unit of Oral Health Sciences, University of Oulu, Oulu, Finland.
Virve
V
Pääkkönen
Research Unit of Oral Health Sciences, University of Oulu, Oulu, Finland; Medical Research Center, Oulu University Hospital and University of Oulu, Finland; Department of Oral and Maxillofacial Diseases, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
Leo
L
Tjäderhane
Department of Cariology, Faculty of Odontology, Malmö University, Sweden.
Dan
D
Ericson
D019342:Acetic Acid / Q000737:chemistry; D000293:Adolescent; D000328:Adult; D003094:Collagen / Q000378:metabolism*; D003731:Dental Caries / Q000201:enzymology / Q000378:metabolism; D003804:Dentin / Q000737:chemistry / Q000378:metabolism*; D003956:Dialysis / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D020784:Matrix Metalloproteinase 8 / Q000378:metabolism*; D017001:Tooth Demineralization / Q000378:metabolism; D055815:Young Adult
C093073:Demineralized Dentin Matrix; D003094:Collagen; D004492:Edetic Acid; C488762:MMP8 protein, human; D020784:Matrix Metalloproteinase 8; D019342:Acetic Acid
Acetic acid; Collagen; Edetic acid; Matrix metalloproteinase; Tooth demineralization