Characteristics And Roles Of Hertwig's Epithelial Root Sheath And Epithelial Cell Rests Of Malassez During Tooth Development

PURPOSE: To observe the morphology, apoptosis, proliferation and distribution of Hertwig's epithelial root sheath (HERS) and epithelial cell rests of Malassez(ECRM) during tooth development, and their expressions of bone-related noncollagenous glycoproteins.METHODS: Postnatal BALB/c mice (Research Animal Center Shandong University) of different development stages were used. Anesthetized animals were sacrificed by vascular perfusion through the left ventricle and into the aorta. The vasculature was pre-rinsed with 0.9% physiological saline for 30s followed by perfusion for 30min with 4% paraformaldehyde (pH 7.0). The mandible were dissected and immersed in the same fixative solution for an additional 24h at 4℃. After decalcificated with 10% EDTA for 1-2 month, dehydrated through graded ethanol, cleared with xylene and embedded in paraffin, and 5 μm thick continuous slices were made. The sections were stained with hematoxylin and eosin (H.E.) and observed by light microscopy.Cytokeratinl4(CK14) antibody was applied as a marker to trace the morphology and distribution of HERS and ECRM cells. The morphology and distribution of HERS and ECRM were examined by light and transmission electron microscopy (TEM). For further study their biologic chrarcteristics, apoptosis was identified by the terminal deoxy-transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method. PV two-step immunohistochemical method was used to detect the expression of bcl-2 protein, PCNA (proliferating cell nuclear antigen), BSP (bone sialoprotein) and OPN (osteopontin) in HERS and ECRM.The number of ECRM cells in pre-OFP, OFP (occlusion found period) and post-OFP were quantified using Student-Newman-Keuls (SNK). The criteria for statistical significance were accepted at the probability level p < 0.05. RESULTS: After the completion of crown formation, the inner and outer enamel epithelium fused below the level of crown cervical enamel to produce a bilayered epithelial sheath termed HERS, which positively expressed PCNA but negatively Bcl-2. Immediately after formation of predentin, HERS postively expressed TUNEL and then disintegrated. Cementoblasts could be found beside these disintegrated HERS. Later, some cells migrated into the periodontal ligament and aggregated to form ECRM cell clusters. However, several cells incorporated in the cementum, positively expressed CK14, which hinted their epithelial origin.During the fuse of inner and outer enamel epithelium, some cells, which displayed the cytologic features of protein synthesis and secret, could be found between the two epithelium cells. Desmosomal cell junctions were observed. The positive expressions of OPN were observed at the beginning of HERS. An accumulation of OPN could also be observed at dentin-cementum junction during cementogenesis. But no positive BSP signals were seen at onset of HERS.ECRM experienced instinct morphological changes during tooth emergence and occlusal function. They were observed as network of epithelial cells labeled by CK14, especially in furcation level regions of mouse molars and active cell proliferation during occlusion found period. Cell apoptosis was observed in many ECRM by transmission electron microscopy during late stage of the progess. The number of ECRM in furcation level regions of mandibular 1^st molars increased (P < 0. 01) in OFP comparing with that in pre-OFP.CONCLUSION: At onset of HERS, the inner enamel epithelium, outer enamel epithelium embrace several cells with developed cytoplasmic organelles, which may derive from stratum intermedium or stellate reticulum, and form a structure like enamel organ, which may be associated with initial cementogenesis. OPN, the non collagenous matrix proteins of cementum, may also take part in the formation of the dentin-cementum junction. Apoptosis of HERS cells may signal the development of cementum, and some HERS cells maybe directly participate in the progress.ECRM may not only an accidental left-over of early embryonic development but rather play significant roles in OFP, which may hinted it different roles from HERS.