Expression Of PLAP-1 In Normal Periodontal Tissues And Effect On Differentiation Of ST2 Cells Into Osteoblasts

Background and Objectives:The periodontal ligament is a connective tissue interposed between the roots of teeth and the inner wall of the alveolar bone socket. And it has been revealed that the periodontal ligament possesses multipotential mesenchymal stem cells that can differentiate into mineralized tissue-forming cells such as osteoblasts and cementoblasts. However, the periodontal ligament is never ossified in vivo under normal circumstances. This suggests that some mechanisms exist to constitutively prevent unorchestrated osteogenesis and cementogenesis by the PDLC.A novel gene named PLAP-1 was discovered in the gene expression profile of the human periodontal ligament. PLAP-1, as a new member of extracellular matrix proteoglycan, belongs to the class I small leucine-rich repeat proteoglycan(SLRP) family. Some findings are supported that PLAP-1 suppresses chondrogenesis by inhibiting TGF-βfunction through a direct interaction and associates with osteoarthritis, rheumatoid arthritis and lumbar-disc degeneration. And it plays a specific role in the periodontal ligament as a negative regulator of cytodifferentiation and mineralization.The ST2 cells are mesenchymal cells derived from bone marrow matrix in BC8 mouse, with multiple differentiation potential. It can be differentiate into osteoblast cells in the osteoblast medium(OBM). Therefore, it is often used as a cellular model for researching the differentiation of osteogenic cells. There is litter research on the expression of PLAP-1 in normal periodontal tissues and the function of osteoblast diffenentiation. So, the aim of this paper was to study the expression and transcription of PLAP-1 in the periodontal tissues and PDLC by immunohistochemistry and reverse transcription-polymerase chain reaction. Meanwhile, we transfected PLAP-1 into ST2 cells by retroviral system, to determine the effect of PLAP-1 on differentiation of ST2 cells into osteoblasts, and to offer some cellular and molecular information for homeostasis of periodontal tissues and periodontitis.Materials and Methods:1. Expression of PLAP-1 in normal periodontal tissues and its cells in ratMandibles and maxillaries including the molars were removed from normal SD rat and 5μm sections were prepared. Immunohistochemical technique was adopted to determine the tissue distribution of PLAP-1 in normal periodontal tissues. Then molars were extracted from mandibles and maxillaries of rat under stereomicroscope and periodontal ligament tissue was removed from the mid-third of the root. Primary PDLC were obtained by the method of tissue culture. Subsequently, experiments were carried out with cells from the third passages. RT-PCR and immunocytochemistry were used to detect the expression of PLAP-1 mRNA and protein in PDLC.2. Effect of overexpression of PLAP-1 on differentiation of ST2 cells into osteoblastsPLAP-1 recombinant retroviral plasmid vector pBABE-hygro-PLAP-1 was constructed and transfected into the retroviral packaging cells 293T by LipofectamineTM 2000 to produce PLAP-1 retrovirus. The medium from packaging cells including retrovirus was added to infect ST2 cells and chosen by Hygromycin B to gain the stably overexpressing ST2 cells. RT-PCR and immunocytochemistry were adopted to determine protein expression and mRNA transcription of PLAP-1 in ST2 cells. And mineralization assay was adopted to determine the effect of overexpression of PLAP-1 on differentiation in ST2 cells. Results:1. Expression of PLAP-1 in normal periodontal tissues and its cells in ratPLAP-1 was expressed positively in the periodontal ligament while negatively in the other periodontal tissues cementum, alveolar bone and gingival tissues. On cells level, cultured periodontal ligament cells expressed positively both in its protein and mRNA according to immunocytochemistry and RT-PCR study.2. Overexpression of PLAP-1 in ST2 cells suppresses mineralizationThe construction of the recombinant eukaryotic expression plasmid pBABE-hygro-PLAP-1 was confirmed through restriction enzyme maping analysis and DNA sequencing. PLAP-1 retrovirus packaged by 293T was added to infect ST2 cells and antibiotic selection 10 days to obtain stably overexpressing PLAP-1 cells. RT-PCR and immunocytochemistry analysis confirmed that the transfected cells were overpressing PLAP-1 comparing with that in the control cells. ST2 stably overexpressing PLAP-1 was successfully constructed by retroviral system. Mineralization assay showed that the mineral nodule formed in ST2 cells overexpressing PLAP-1 was significantly less compared with that in wild ST2 cells(P<0.05).Conclusions:1. PLAP-1 is express specifically and predominantly in the periodontal ligament. It indicates that PLAP-1 plays an important role in physiological functions of periodontal ligament.2. By retroviral gene transfer approach, overexpressing PLAP-1 may negatively regulate the differentiation of osteoblasts.