The Cellular Biology Characteristic Of Rat Enamel Organ Epithelium Under Three-dimensional Cell Culture System

The ameloblast plays a very important role during the development of tooth. The ameloblast not only secrets enamel matrix to form the enamel, but also controls the differenciation and secretion of matrix of the odontoblast. There are a lot of reason that can cause the ameloblast differentiation abnormal,such as envinronment factor,dystrophia,trauma, or a variety of genetic disorders. The ameloblast differentiation abnormal can cause a lot of disease, such as mottledenamel, tetracycline discoloration, enamel hypoplasia, dentinogenesis imperfect, et al. Selection and culture of ameloblast lineage cell within the enamel organ epithelium are necessary for studies of ameloblasts function, pathology and enamel formation.The ameloblast is a kind of differenciated cells. It degenerated into induced enamel epithelium when the crown has formed, and losed when the tooth eruption.So it is very difficult to get and culture in vitro. Until now, two-dimensional monolayer cell culture method is the most common method for the research of cellular biology and molecular biology. There are a lot of methods of ameloblast lineage cell culture in vitro, but all are based on the two-dimensional cell culture system. There are a lot of significant differences on the gene expression, cellular secretion and cell functioned activities of cells cultured in the two-dimensional culture system and in the three-dimensional culture system. It is the most important that how to obtain the cell culture method which can mimic the in-vivo environments and facilitate observing the interactions between epithelial cells and mesenchyme cells.The objective of this study were to separate and purify the dental EOEs; to identify whether RCCS can be used for culturing the dental EOEs; to observe the differences of cell proliferation and differentiation characteristics of dental EOEs cultured in RCCS and in the traditional cell culture system; to observe the differences of cell proliferation and differentiation characteristics of dental EOEs in RCCS from in the traditional cell culture system;to study the effect of dental papilla cells on the cell proliferation, cell viability and the differentiate of dental EOE in the special three-dimensional cell culture system-RCCS; to investigate the identify and self-assemble of dental EOEs and papilla cells in ECM. The present study consists of two parts:Part one: the cellular biology charactorastic of the rat enamel organ epithelium under the three dimensional cell culture systemIn order to study the effect of three-dimensional cell culture system on the characteristic of ameloblast cellular biology, we culture the rat enamel organ epithelium in the RCCS, and analysis the effect of three-imensional culture and the interaction of mesenchyme on the EOE, respectively.Firstly, we used dispasnase to separate the papilla from the enamel organ, and used the different speed of epithelium and mesenchyme to stick to the plate to purify enamel organ epithelium, and culture in DMEM/F12 medium. The post-natal 4 day rats enamel organ epithelium purificated rapidly and present cobblestone morphology, cellular connection is conjected tightly, epithelium grow like lamellar layer Several stellate cells grow between epithelium islands. Immuocytochemistry analysis reveals cobblestone morphology cells are positive in CK14 staining, but negative in vimentin staining. The pnd-4 rat pups were used to do the next study.Secondly, the rat enamel organ cells were cultured in RCCS three-dimensional culture system. To scale-up anchorage-depended cell in RCCS bioreactor must use the microcarrier as the scaffold. The microcarrier can provide the nessessary growth areas for anchorage-depended cells. The RCCS is suitable for differentiated cell culture, particularly for cells with an epithelial-like morphology. The concentration of microcarriers and EOE density were adjusted by observing the cell growth by phase-contrast microscopy, obtain the key techniques of EOE culture in RCCS. We find that the cell can attached on the surface of the microcarriers and form cell/microcarriers three-dimensional aggregation ; cytodex-3 can provide efficient cell growth surface for EOE at the concentration of 1mg/mL; the lowest inoculation indensity of EOE is 1x105cells/mL。By comparing cellular growth curve in RCCS and dynamic culture condition, we find that RCCS culture system can significantly increase the number of enamel organ epitheliums, 5x106cells/mL and 1x106cells/mL, for RCCS and dynamic traditional culture, respectively. Much higher inoculation density was required in RCCS than in dynamic traditional culture condition did. cell can't amplify in the condition of lowest inoculation indensity (0.5x105cells/mL). We also find that in RCCS the cellular activity is higher and EOE characteristic functionalized protein (ameloblastin protein) expression is higher than the dynamic culture group. We also confirm this result by analyzing the mRNA of ameloblastin and amelogenin. Throughout the experiments, glucose was staying within the accepted physiological range. This suggests that glucose is not a limiting factor in these cultures.Lastly, the dental development is the result of epithelium-mesenchyme interaction. The in vitro co-culture system is the helpful tool to study the mechanisms of epithelium-mesenchyme interaction. RCCS is the only equipment which can provide co-culture study until now. In this part, we co-culture rat enamel organ epitheliums with dental papilla cells in RCCS, contract with the dynamic traditional culture systems. The results reveal that RCCS system can improve the proliferation of EOE at the finial density of 8.6×10⁶cells/mL and 3.14×10⁶cells/mL, for RCCS and dynamic traditional culture, respectively.It can significantly improve the EOE cellular growth speed and increase folds in the condition of co-culture with dental papilla, the concentration of microcarriers needed in RCCS co-culture system was 3mg/mL. The scanning electron microscropy analysis results show that there are a lot of cells/beads aggregations, and the cell can growth three dimensionaly. The positive cellular number of ameloblastin protein fluorescent staining is higher than the dynamic culture condition. The RT-PCR result showed the similar trend of marker protein mRNA expression :the expression of p75NGFR reach the maximum on day 5, but the mRNA expression of ameloblastin and amelogenin increased constantly, reaching the maximum on days 7. We can monitor the glucose level,LDH release rate,the lactic acid level,pO₂,pCO₂,pH changes in this three-dimensional culture system by biochemistry and Blood Gas Analyzer, the results show that the level of LDH is the low condition all the time except for lag phase. Although the level of glucose, PH value and oxygen pressure is lower than the dynamic situation, but still in the physical range. There was no significant difference of biochemistry and Blood Gas Analysis between co-culture and EOE cultured in RCCS system.In short, we have grasped the key culture techniques of rat enamel organ epithelium in vitro RCCS successfully, and establish the study model of rat enamel organ three-dimensional culture system, and have studied the growth characteristic of rat EOE in this culture system, It is the experimental basis for further study.Part two: The identification and self-assemble of dissociated dental germ cells in the Collagen/Matrigel ScaffoldTo imitate the in vivo microenvironment of dental germ cells, the self-made artificial ECM was used to culture the dissociated dental germ cells three dimensionally and to study the identification and self-assemble of dissociated dental germ cells and dental papilla cells, and also the biological behaviors.Firstly, we prepared artificial ECM protein gel by mixing Matrigel and type I collagen solution, which was the solution of rat tail tendon with 0.1% acetic acid. and have gotten the key techniques of preparing artificial ECM protein gel with Matrigel and type I collagen by adjusting pH. Secondly, rat tooth germ cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During the days of in vitro culture, the dental constructs were observed under microscope and analyzed using histological and immunohistochemistry examination. We find that Matrigel can improve the proliferation of EOE. The EOEs and mesenchyme cells grown healthy in artificial ECM protein gel with the best mixed ratio of 9 type I collagen to 1 Matrigel.Secondly,cell density is vital to determine the intensity of contraction. At high cell density, the constructs break down. Because of intensively stretching the constructs break and center cellular necrosis. The optimal concentration was about 5×10⁶cells/ml. With this concentration, most structures containing *and* structures formed.There are two growth type of EOE in artificial ECM gel: one type is the EOE creped to the surface of gel to form epithelium layer and proliferated at a point to form a deepen epithelium strip into gel and mesenchyme cells surrounding them, this structure is similar to tooth bud; the other type is CK 14 positive EOE cells proliferated inside the ECM and formed epithelial pearl-like structures, which vimentin positive dental papilla cell surrounding these epithelial pearl-like structures. These structures are similar to enamel organ and dental papilla tissue.Dissociated enamel organ cells and dental papilla cells keep the primary biological behaviors to form similar architecture in different dental germ periods, have tendency to form tooth in vitro three-dimensional culture condition. We need to optimize culture conditions continually as the limitation of growth environment provided by in vitro three-dimensional culture system.In short, the results revealed that RCCS and artificial ECM is the helpful tool for studying the interaction between dental germ cells and mesenchyme cells in three-dimensional culture condition. This research obtains the key techniques of enamel organ cells- mesenchyme cells, enamel organ endothelial cells in RCCS. This system can amplify high quality enamel organ cells and the research tool for studying interaction between dental germ cells and mesenchyme cells in three-dimensional culture condition. We have exploratory study biological characteristic and interaction recognition, self-assemble of epithelium cellula intersitialis. It can provide biological theory basis for dental regeneration.