Study Of Human Periodontal Fibroblasts Induced Into Junctional Epithelium-Like Cells

Objective:As a special epithelial structure, junctional epithelium (JE) attaches to dental cervix, it connectes connective tissues with the tooth surface and forms dento-gingival junction. JE derived from reduced dental epithelium has no stratum corneum and epithelium pegs. Due to the weakness of physiology, JE was destroyed at first when periodentitis occurred, which leaded to periodonal pocket formed and alveolar bone resorbed. As a result, periodontitis developed quickly and actively, which made the serious loss of chewing function. However, the repair of dento-gingival junction was very slowly even after the lesions repaired, reconstruction of JE structure was difficult. So, it is the key and emphasis in regeneration of JE for rebuilding periodontal functional structure. This was to study the morphological and immunohistomical characteristics of the junctional epithelium-like cells, which was induced by human peridontal fibroblasts, and grew on acellular dermal matrix (ADM) and small intestinal submucosa (SIS) scaffolds. The results could provide theory basis and laboratory data of epithelium source of dento-gingival junction.Periodontal membrane, also called periodontal ligament, is a sort of connective tissue between teeth root and alveolar bones, which plays an important role in periodontal support for the teeth. Periodontal membrane is mainly composed of fibroblasts. Previous studies have proved that periodontal fibroblasts possess the properties of stem cells, periodontal tissue has strong capability to repair, immigration and differentiation from different subpopulation cells. This study was to reveal that human periodontal fibroblasts if possess the properties of multi-lineage differentiation as stem cells by inducing into junctional epithelium-like cells.With the development of tissue engineering, the important study is to select appropriate scaffold. At present, many kinds of scaffold are used, such as small intestinal submucosa (SIS), guided tissue regeneration collagen (GTRC), acellular dermal matrix (ADM) and nanometer calcium hydroxyapatite. These scaffolds are biocompatible, high porous and biodegradable. In this study, ADM and SIS were chosen to be the scaffold tissues on which junctional epithelium-cells cultured, in order to discuss the cell adhesion on the scaffold and tissue compatibility, the immunocharacterization and ultrastrcture of junctional epithelium-cells was observed.Methods:1 Culture and identification of human periodontal fibroblastsNormal human premolars extracted for orthodontic therapy were collected from patients (12~30 yrs of age) in Hospital of Stomatology, Hebei Medical University. Periodontal ligament was obtained from the middle 1/3 of the root under sterile conditions. Periodontal ligament cells were cultured in DMEM-LOW GLUCOSE contained 20% FBS by using traditional method of primary culture. The third generation cells were identified by Immunohistochemical stain against Vimentin and CK antibodies respectively.2 Induced and identified of human periodontal fibroblastsThe third generation human periodontal fibroblasts were cultured in K-SFM for 7~10 days. The cells induced were identified by Immunohistochemical stain against CK antibody.3 Preparation for the scaffold of small intestinal submucosa (SIS)Serosa and muscle of the jejunum were removed by gauze. Then, the jejunum was soaked in PBS contained penicillin and streptomycin for 3 h, 0.25 % trypsin for 24 h , rinsed by distilled water, soaked in 0.5% SDS for 24 h, rinsed by distilled water, sterilizated in 20% ethanol containedand 0.1% peracetic acid for 10 h, freeze-dried disinfected for use.4 Human periodontal fibroblasts induced on ADM and SIS scaffcold.Human periodontal fibroblasts planted on ADM and SIS scaffold were cultured in K-SFM for 3~5 days. Immunohistochemical identification against CK antibody and electron scanning microscope (SEM) was used respectively to observe the tissues which the human periodontal fibroblasts induced and grew on ADM and SIS scaffcold.Results:1 Culture and identification of human periodontal fibroblastsSpindle cells with round or ellipse nucleus containing 1~3 nucleolus were observed in primary culture. Immunohistochemical stain against Vimentin antibody was positive and CK antibody was negative in the cytoplasms of human periodontal fibroblasts.2 Induced of human periodontal fibroblastsImmunohistochemical stain of CK was positive in the cytoplasms of the junctional epithelium-like cells after induced, which showed the immunohistological feature of epithelium.3 Observation of small intestinal submucosa (SIS) by electron microscope (SEM)Observation by SEM, SIS used as a scaffold consisted of fiber bundles except any cell compound.4 Observation and identification of human periodontal fibroblasts on scaffoldsThe Juntional epithelium-like cells on scaffolds showed CK positive immunohistochemical reaction in the cytoplasms. The results of ESM showed that the Juntional epithelium-like cells on scaffolds grew flaky, polygon and flat, with round nucleus containing 1~2 nucleolus, and the Juntional epithelium-like structure was formed.Conclusions:1 Human periodontal fibroblasts are potential ability to differentiate to junctional epithelium-like cells.2 Human periodontal fibroblasts could grow on the different scaffolds to form juntional epithelium tissue.