The Role Of Periodontal Ligament Stem Cells In The Physiological Root Resorption Of Primary Teeth

Toot root resorption is the breakdown or destruction, and subsequent loss of the root structure of a tooth. There is no uniform classification for root resorption. Andreasen et al divided the root resorption into surface resorption, inflammatory resorption and replacement resorption. According to the site of root resorption, Tronstad dividied the root resorption into internal absorption and external absorption. According to etiology, Fuss attributed the root resorption to endodontic infection, periodontal infection, orthodontic treatment caused root resorption and adhesive absorption caused by impacted tooth or tumor pressure. Based on pathophysiology, root resorption can be divided into physiological root resorption and pathological root resorption. Physiological root resorption of primary teeth is a natual and physiolgical phenomenon, and the primary teeth are the only hard tissue which could be absorbed and disappeared physiologically.The exact mechanisms about tooth root absorption are largely unknown. Some scholars considered that it might be relate to the eruption pressure of succeeding permanent teeth, inflammatory environment or occlusal trauma. However all of those considerations are still on the exploratory stage. Since mesenchymal stem cells and the microenvironment paly a pivotal role in maintaining the normal development of bone and teeth, studying the role of stem cells in the physiologic root resorption of primary teeth might give a better understanding to this wonderful physiological processes.In this study, we isolate, culture and identificate the periodontal ligament stem cells (PDLSCs) from unabsorbed, moderate and severe absorption stage of primary teeth for the first time, and the PDLSCs isolated from healthy permanent teeth were used as control. Gene expressions of PDLSCs, including osteogenesis related gene Runx-2, ALP and OPG, osteoclastogensis related gene RANKL and inflammation related gene IL-1, IL-6 and TNF-αin all these three stages of primary teeth and healthy permanent teeth were examined by Quantitative Real-time Polymerase Chain Reaction and Western blot assay, to explore the role of PDLSCs in the physiologic root resorption.Part one:The study on the biological behaviors of PDLSCs derived from primary teeth and permanent teethPDLSCs from unabsorbed, moderate absorption stage and severe absorption stage of primary teeth were isolated, cultured and identified. PDLSCs isolated from healthy permanent teeth were used as control. Cells were identified by the self-renewal, differentiation experiments and microenvironment flow cytometry. The results showed that the cell morphology were similar among the PDLSCs from various stages of primary teeth and permanent teeth. All of them presented spindle-shaped, radial growth and oval nucleus located in the central cytoplasm; MTT and clone formation rate assay showed that PDLSCs from primary teeth have higher vitality than PDLSCs from permanent teeth. PDLSCs from moderate and severe absorption stage of primary teeth presented higher vitality than other stages; PDLSCs from each group have the ablility of the osteogenesis and adipogenic potential, while PDLSCs from the moderate and severe absorption stage of primary teeth have stronger osteogenesis and adipogenic capacity than those of the other stages; flow cytometry results showed the cells were positive for CD146 and CD90, markers of early mesenchymal stem cells , but were negative for markers related to hematopoiesis. Those results suggest that the PDLSCs from primary teeth have stronger self renewal and multi-lineage differentiation capacity than those from the permanent teeth.Part two: The expression of inflammatary, osteobalsts and osteoclasts related factors among various PDLSCsTo explore the expression change of inflammation, osteoblasts and osteoclasts related factors in PDLSCs from four stages of root resorption. PDLSCs from unabsorbed, moderate and severe absorption stage of primary teeth were isolated, cultured and identificated. PDLSCs isolated from healthy permanent teeth were used as control. The expressions of osteogenesis related genes such as RUNX-2 and ALP, and inflammation related genes were up-regulated gradually. Interestingly, osteoclastogensis related gene RANKL also showed up-regulation during this process, On the contrary, the osteogenesis gene OPG was down-regulated, which was different from other osteogensis gene expression. Accordingly, the ability of osteoclastogensis/osteogensis, indicated by the ratio of RANKL/OPG, was also up-regulated. In the process of physical root resorption in primary teeth,IL-1β, IL-6 and TNF-αmight directly stimulate osteoclastogenesis or act synergistically with RANKL/RANK/OPG system to stimulate the formation of osteoclasts, resulting in efficient fusion of preosteoclast cells. On the other hand, transcription factor RUNX2 may be involved in the regulation of classic RANKL/RANK/OPG system to induce the expression of RANKL,and inhibit the expression of OPG, thereby promoting differentiation and maturation of osteoclasts.