Analysis of proteome and transcriptome expressions in periodontal ligament tissue of preserved teeth for replantation

Abstract

Discovering the influencing factors on the result of delayed replantation will contribute to reattach extracted tooth to its alveolar socket and save natural teeth. As the first step of investigation work, it is meaningful to figure out the change of components such as adhesion molecules in periodontal ligament tissue. Therefore, the purpose of this study was to investigate and compare the expressions of proteome and transcriptome in periodontal ligament tissue of extracted and stored teeth under various conditions, and to find possible contributing components to reattachment and regeneration of periodontal ligament.Volunteers were recruited from patients who were scheduled to have 4 premolars extracted in the Department of Orthodontics. Extracted teeth were immersed into storage solution composed of 1.0 L Hartmann’s solution and 1.0 g of Flumarin, and instantly divided to 4 groups – 1) Immediate sampling, 2)1 week storage and sampling, 3) 2 week storage and sampling and 4) 1 week dry storage under room temperature and sampling. Periodontal ligament tissues were collected from the middle-third area of the root with periodontal curette, put into a 1.5 ml tube and stored in liquid nitrogen. Two-dimensional electrophoresis was carried out. The gel image was scanned in a Bio-Rad densitometer and analyzed with Image Master Platinum 5.0 image analysis program. The protein spots that sustained under hypothermic storing solution and decreased under dry storage were firstly selected and identified with MALDI-TOF MS (Matrix-Assistant Laser Desorption and Ionization Time-of-Flight Mass Spectrometry). Secondly, the adjacent spots around an interesting protein spot were additionally analyzed to confirm the protein’s isoforms. The acquired data were searched in Mascot Database of human protein. One way analysis of variance (ANOVA) was applied to compare the protein quantity between 4 groups and Bonferroni test was used in the post hoc comparisons.Twenty seven spots were firstly selected by suggested conditions. Vimentin was identified in 11 spots and located closely in left middle area on 2-DE gels. Then eight spots around 11 Vimentin spots were secondly analyzed. Five spots were identified as Vimentin. Total 16 Vimentin spots showed gradual decreasing from immediate groups to 2 week stored groups and rapid decreasing to dry groups in protein quantity. Only dry group was significantly different from immediate group.Three spots were identified as type VI collagen and located upper left area on 2-DE gels.Immediate sampling group and 1 week stored group were analyzed and compared using RNeasy mini kit. PCR array for human extracellular matrix and adhesion molecules revealed that LAMA1, CTGF and THBS1 were significantly over-expressed in 1 week stored group over immediate group, and MMP9, SPP1 and ITGB3 were significantly under-expressed in 1 week stored group over immediate group.Vimentin and type VI collagen which sustained in stored group can contribute to reattachment and regeneration in periodontal ligament with regard to collagen production. Laminin, CTGF and thrombospondin which induce tissue proliferation were up-regulated in 1 week stored group, and matrix metallopeptidase, secreted phosphoprotein and integrin which are known to be related to tissue degradation were down-regulated in 1 week stored group. These proteome and transcriptome could work in regeneration of periodontal ligament and further research to validate their role and function should be done.