Comparative Analysis of Gene Expression between Cementum and Periodontal Ligament

Objectives: Cementum and periodontal ligament (PDL) play important roles to maintain masticatory function; however, specific markers for cementum or PDL have not been identified. Consequently, molecular mechanism of periodontal tissue development, homeostasis and regeneration poorly reported. In previous studies cementum or PDL tissue, obtained by manual curettage, has been examined, resulting in difficulties isolating pure cementum or PDL tissue and in less possibility finding specific markers. To address this issue, we have tried to establish an experimental system to compare gene expression in cementoblasts with in PDL cells.

Methods: Laser capture microdissection (LCM) was employed to isolate cementoblasts and PDL cells from undecalcified frozen sections of murine mandible and to obtain RNA in good quality for subsequent genetic analysis. Over 500 cementoblasts and PDL cells were separately laser captured under microscopy. Gene expression levels between cementoblasts and PDL cells were compared by GeneChip that contained 40,000 arrays. The primer sets of cementum-positive and PDL-negative genes were serially designed and expression of the cementum-positive and PDL-negative genes in RNA samples obtained from mandibles and femur were analyzed by quantitative RT-PCR.

Results: Bioanalyzer detected peaks of 18s and 28s rRNA both in the laser-dissected cementoblasts and in PDL cells, suggesting the sufficient quality of RNA. A comparative analysis of mRNA expression by GeneChip showed that about 2,000 genes were differentially expressed between cementoblasts and PDL cells. Several genes of cementum-positive and PDL-negative were exceedingly expressed in the mandible than in the femur. Those results suggested that a few of them might be possibly specific markers for the cementoblast.

Conclusion: We established the novel experimental system to isolate target tissues from single cells in undecalcified frozen sections and to obtain intact RNA. These sequential methods could be useful for genetic studies, especially, to explore tissue-specific factors.