Effect Of PLAP-1on Bone Regeneration In Wistar Rat Skull Defect Model

Background and Objectives:The periodontal ligament (PDL) is a dense connective tissue located between the tooth root and alveolar bone. The cells in PDL have been revealed to be multipotent cells that have the ability to differentiate into osteoblasts or cementoblasts depending on the microenvironment.However, under normal physiological conditions, the periodontal ligament is a tough and resilient organization, never ossified all life long.It has been found that periodontal ligament-associated protein-1(PLAP-1) plays a crucial role for this homeostasis of periodontal tissues. PLAP-1was discovered and defined in the gene expression profile of the human periodontal ligament. In the process of differentiation and mineralization of the periodontal tissue, PLAP-1as a negative regulator prevent it excessively osteogenesis with other growth factors. And in the pathogenesis of osteoaritis, rheumatoid arthritis and lumbar-disc degeneration, PLAP-1plays a same role of suppresses chondrogenesis.Bone marrow stem cells (BMSCs) have multi-lineage differentiation ability. They can differentiate into fibroblasts, osteoblasts, cementoblast and the others depending on different inducing conditions. It has been confirmed that co-cultured with periodontal firbroblasts in vitro study, BMSCs exhibited the characteristics of periodontal fibroblasts. BMSCs have a good proliferate capacity and be easy to obtain. So, BMSCs can be used as seed cells for studies of periodontal tissue regeneration replace the periodontal ligament fibroblasts.Rat critical size defect model is a classical model for evaluation of bone restorative effects in bone tissue engineering. The morphology and embryonic origin of skull are similar to maxillofacial of most of the mammals:skull and jaw are single membrane bone (except for the mandible, sphenoid wing). The skull and mandible are constituted by two layers of cortical bone within cancellous bone in the anatomical structure aspect. And in the physiological, the cortical bone of skull is similar to the atrophy mandible. Therefore, this model is the ideal replacement of the mandible defect model.So far, the researches of PLAP-1are limited to in vitro; this experiment is the first attempt to study PLAP-1in vivo. First, constructed the stably overexpressing PLAP-1gene in Rat-BMSCs; Real-time quantitative PCR and immunocytochemistry were adopted to determine mRNA transcription and protein expression of PLAP-1in Rat-BMSCs, and a variety of osteogenic marker genes were detected. Second, the overexpressing PLAP-1Rat-BMSCs were transplanted into the rat skull critical size defect, after8weeks, the effects of repairs were observed expecting offer some cellular and molecular information for homeostasis of periodontal tissues and periodontitis.Materials and Methods:1. Effect of overexpression of PLAP-1on differentiation of Rat-BMSCs into osteoblastsPLAP-1recombinant retro viral plasmid vector pBABE-hygro-PLAP-1was extracted and transfected into the retroviral packaging cells293T by LipofectamineTM2000to produce PLAP-1retro virus. The medium from packaging cells including PLAP-1retro virus was added to infect Rat-BMSCs cells and chosen by Hygromycin B to gain the stably overexpressing PLAP-1Rat-BMSCs. Real time-qPCR and immunocytochemistry were adopted to determine mRNA transcription and protein expression of PLAP-1in Rat-BMSCs. After a week of mineralization, a variety of osteogenic marker genes were detected.2. Animal experimental study of effects of PLAP-1on Skull Defect of Wistar Rat. Grouped into the Collagen carrier-overexpressing pBABE-hygro-PLAP-1Rat-BMSCs complex; the Collagen carrier-overexpressing pBABE-hygro- Rat-BMSCs complex; the Collagen carrier-Rat-BMSCs complex; the Collagen carrier and blank control group.Prepared collagen carrier by cattle â…  collagen, cultured cells, and they were co-cultured. Collagen carrier-cells complex were transplanted into the Wistar Rat skull critical size defect. At8weeks, the specimens were obtained. The effects of skull defect of Wistar Rat were evaluated through generally observed of the specimen, the X-ray examination, HE staining, immunohistochemical staining, Masson staining and Kossa staining etc in paraffin and hard tissue slice.Results:1. Effect of overexpression of PLAP-1on differentiation of Rat-BMSCs into osteoblastsPLAP-1retrovirus packaged by293T was added to infect Rat-BMSCs and antibiotic selection10days to obtain stably overexpressing PLAP-1cells. Real time-qPCR and immunocytochemistry analysis confirmed that the transfected cells were overpressing PLAP-1comparing with that in the control cells. Rat-BMSCs stably overexpressing PLAP-1were successfully constructed by retroviral system. The level of expression of various ostegenic genes of overpressing pBABE-hygro-P LAP-1-Rat-BMSCs was lower than the overexpressing pBABE-hygro-Rat-BMSCs and Rat-BMSCs.2. Animal experimental study of effects of PLAP-1on Skull Defect of Wistar Rat.The repair effect of the group of Collagen carrier-overexpressing pBABE-hygro-PLAP-1Rat-BMSCs complex was significantly worse than the groups of the Collagen carrier-overexpressing pBABE-hygro-Rat-BMSCs complex and the Collagen carrier-Rat-BMSCs complex through generally observed of the specimen, the X-ray examination, HE staining, immunohistochemical staining, Masson staining and Kossa staining.Conclusions:1. The levels of expression of various ostegenic marker genes of overpressing PLAP-1-Rat-BMSCs are significantly lower than the control cells.2. Overexpression of PLAP-1inhibits the repair effects of Wistar Rat skull defects.