Study On Characterization Of Tooth Root Developing Stage Related Mesenchyma Stem Cells

Tooth crown engineering has got great progress in recent years, but nowadays, the major problem in tooth tissue engineering is the complete tooth regeneration, especially the bioactive tooth root reconstruction. How to build the bioactive-root is a "bottleneck" of tooth tissue engineering, which directly influenced dental tissue engineering and eventually determines the direction of the related studies. Seed cells are acting as one of the key factors in tooth tissue engineering. Seed cells applied in traditional root/periodontal tissue engineering mainly comes from the adult stem cells. In view of its characteristics such as absence of ethical problem, capability of in vitro operation, source richness, easy plasticity and no immune rejection (etc), adult stem cells have become the predominant seed cells for root / periodontal tissue engineering. However, there are series of problems, such as: 1. the source of adult stem cells is not stable or reliable; 2. the number of adult stem cells is very small so that it needs to be expanded on a large scale in vitro first, while it's difficult to maintain their status (of stem cells); 3. the methods which can achieve directional induction of adult stem cells differentiation to form a stable structure is under research. All these disadvantages eventually limit the development of root / periodontal tissue engineering.However, with the emergence of development tissue engineering, new ideas and methods have been provided for the root / periodontal tissue engineering. Compared with crown formation, the development of R/PT involves a complicated sequence of interaction and participation of multiple cell types. It has been proved that the apical end of developing root which consists of dental follicle, dental papilla as well as Hertwig's epithelial root sheath (HERS) is the"growth center"of tooth root and contains rich source of stem cells for R/PT regeneration, and with epithelium- mesenchyma interaction, is capable to form R/PT regeneration independently. Then we hypothesize that mesenchyma stem cells from DAC are heterology,which have the potential ability to regenerate root/periodontal complex structures as a new kind of seed cells. Therefore, this study focuses on characteristics of the mesenchyma stem cells at tooth root developing stage and the evaluation of the possibility of being seeds cells.The major findings are as follows:Part I: Identification of the phenotype of DAC-mesenchyma stem cells clone.To evaluate the possibility of DAC- mesenchyma stem cells being seeds cells for R/PT regeneration, we testified the characteristics of DAC-MSCs; the results showed that DAC-MSCs exhibited significant "embryonic" characteristics: such as higher proliferative activity, colony-forming capacity and multilineage differentiation capability in vitro. After transplanted in nude mouse for 8 weeks, DAC-MSCs can regenerate cementum-like structures and dentin-like structures. At the same time, we used limiting dilution assay to screen monoclonal cells from DAC-MSCs and discovered two kinds of phenotype cells: one is osteogenesis type, and the other is fibro singtype. The osteogenesis phenotype monoclonal cells generated major mineralized structures while fibrosing phenotype monoclonal cells generated fibrous structures.The results of this study show that as developing tissues, DAC mesenchyma stem cells still feature unique"embryonic"characteristics and can serve as seed cells for root / periodontal tissue engineering. And it is theoretically feasible to recombine different phenotype monoclonal cells to build a complete reorganization of biological activity of the root / periodontal structure.Part II: Characteristics of periapical follicle stem cells.The aim of this study was to investigate characteristics of periapical follicle stem cells (PAFSCs) isolated from human third molars at the root-developing stage and evaluate the potential usage of these cells for periodontal tissue engineering with human periodontal ligament stem cells (PDLSCs) as control. Putative PAFSCs were isolated and subcultured until 20th passage. Cell characteristics of PAFSCs at early or late passage were evaluated and compared with PDLSCs via a series of histological, cellular and molecular analyses. PAFSCs at early passage possessed crucial stem cell properties and showed a higher proliferation rate and broader differentiation capability than PDLSCs. When transplanted into immunocompromised mice, PAFSCs were able to form cementum/periodontal ligament like complex. During long-term passage, both cell populations underwent a change in morphology and gradually lost their stem cell properties. ALP activity and the expression of mineralization-related genes were markedly declined with an increasing number of passages, which might have caused the loss of in vivo tissue regenerative capacity.Our findings suggest that developing tissue-derived PAFSCs are a distinctive cell population from PDLSCs and might be a novel possible candidate cell source for periodontal tissue engineering. In tooth tissue engineering, seed cells should not be prolonged passaged in vitro to avoid stem cell property changes.