The Study On Biological Behaviors Of Periodontal Ligament Stem Cells Derived From Children With Hypophosphatasia

Hypophosphatasia (HPP) is an inheritable disorder characterized by defective bone and dental mineralization and a deficiency of tissue non-specific alkaline phosphatase (TNAP) activity. The disease is caused by mutations in the liver/bone/ kidney alkaline phosphatase gene (ALPL) encoding TNAP protein. The symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to early tooth loss without bone symptoms. According on clinical manifestation, Hypophosphatasia can be classified into perinatal HPP, infantile HPP, childhood HPP, adult HPP and odontohypophosphatasia. The diagnosis is based on serum alkaline phosphatase assay and mutation analysis of the liver/bone/kidney alkaline phosphatase gene (ALPL). Stem cell biology has become an important field for the understanding of tissue regeneration and implementation of regenerative medicine. Dental stem cells are self-renewal and display multidifferentiation potentiality. In this study, through clinical and laboratory examination combined with pathological examination and electron microscopy analysis of deciduous teeth in one child with hypophosphatasia, we made a preliminary diagnosis to make sure the phenotype of the patient and his symptoms. Periodontal ligament stem cells(PDLSCs)of primary teeth were isolated and cultured. We did some analysis of their biological behaviors. By comparing the biological characteristics of periodontal ligament stem cells from the deciduous teeth between HPP patient and normal children, we aimed to explore the effects of low activity of TNAP on the differentiation of periodontal ligament stem cells and to provide a theoretical basis for the diagnosis and treatment for the disease.Experiment one: The clinical, laboratory and pathological examination of the HPP patientWe managed carefully the clinical and laboratory examinations for the HPP patient. To observe the structure and development of deciduous teeth, we did some analysis on the teeth by pathological methods and electron microscope. The patient showed the poor development of teeth without symptoms of bone. Laboratory tests found that the serum alkaline phosphatase activity of HPP patient was lower than that of health children, and urine PEA testing was positive. Pathological examination and scanning electron microscope results showed that dentin and cementum of HPP patient's deciduous teeth were poorly developed with a low mineralization. So the patient was diagnosed as odontohypophosphatasia.Experiment two: The study on the biological behaviors of PDLSCs derived from normal deciduous teethIn this study, we isolated and cultured periodontal stem cells from deciduous teeth for the first time. By immunocytochemistry test and flow cytometry examination, we identified the origin of these stem cells. We also did some analysis on the proliferation and differentiation abilities of periodontal ligament stem cells derived from deciduous teeth. The results showed that the cells were fibroblast-like and presented with positive expression of mesenchymal surface markers STRO-1, CD146, CD29 and CD90, and negative expression of the hematopoietic cell marker CD34. The proliferation of PDLSCs derived from deciduous teeth was higher than those from permanent teeth. These stem cells could be induced into osteoblast and adipocytes in vitro. These results suggested that stem cells from the periodontal ligament of deciduous teeth were mesenchymal origined. They were highly proliferative and had multiple differentiation capacity.Experiment three: The study on the biological behaviors of PDLSCs derived from HPP patient's deciduous teethBy comparing the biological behaviors of periodontal ligament stem cells from the deciduous teeth of patients and normal children, we aimed to explore the effects of low activity of TNAP on the proliferation and differentiation of periodontal ligament stem cells. The results of our study showed that PDLSCs from HPP patient presented the weaker proliferation and differentiation ability compared with PDLSCs from healthy children. Real-time PCR results showed that the expression of ALP, OCN, CP23 were higher in PDLSCs from HPP patient. But RUNX2 expression was lower compared with normal PDLSCs. Normal PDLSCs from deciduous teeth can form typical PDL-like fibrous tissue in vivo. But PDLSCs from HPP patients can not. These results suggested that low activity of TNAP may play a role in the proliferation and differentiation machanism of stem cells.