Cultivation And Differentiation Of Human Dental Papilla Mesenchyme Cells

The pulp-dentin complexity is main body of tooth and is a key for reconstruction tissue engineering tooth. For this reason, the reconstruction of the pulp- dentin complexity will become the investigative focal point in field of tooth tissue engineering in the future. According to the theories and method of tissue engineering, we study systematically the seed cell and the leg material of reconstruction pulp- dentin complexity, and so on.we expect to to provide the rationale and the experiment technique routes for reconstruction of dental pulp-dentin complexity.Our experimental objective are: 1.To isolate and culture human dental papilla mesenchymal cells (hDPMCs) in vitro, to study cell biology character of hDPMCs and to provide the cell source for next research. 2.To inquiry the influence of growth factor to the amplification and the differentiation ability of hDPMCs in vitro. 3.To study the differentiation mechanism of hDPMCs toward odontoblast. 4.To research the new degradabale bracket material for tissue engineering dens, to inquiry into the growth condition of hDPMCs on the bracket material and to provide the theory system and the experiment methods for thereconstruction of tissue engineering pulp- dentin complexity.The experimental method are: l.To choose spontaneous abortion human embryo of 3-4 months fetal age, to isolate dental papilla from germ, to culture hDPMCs with tissue cake method, to authenticate the hDPMCs cell with the dual dyeing of the vimentin and cytokeratin; To observes the growth property of the cell with inverted microscope; To research extra-cellular matrix forming ability in vitro by immunocytochemistry dyeing of collagen I, fiberonectin, and laminin; To Carry on mineralization ability of hDPMCs by inducing cell to create mineral tubercle that go the calcium salt dyeing. 2. To observe the influence to the hDPMCs amplification and ALP by stimulating the hDPMCs with different concentration IGF-I and bFGF, and to observe the influence of growth factor to hDPMCs propagate and ALP activity at different time of action. 3. To induce hDPMCs toward odontoblast with 100 ug/L IGF-I or 10 ug/L bFGF convenient, by inverted microscope, enzyme chemistry, immunocytochemistry and RT- PCR method, to examines the morphological and functional change of the cells. 4. To make the PDLLA multiporous froth leg material with DL- lactic acid(85%) for tissue engineering pulp-dentin complexity; to observe material pore condition with SEM and fluid substitution method and examine physico-chemical property of material. To use the inverted microscope, SEM, the flow cytometry examine cell amplification situation under the compound culture of hDPMCs and the PDLLA froth leg material in vitro. To implant the hDPMCs froth leg material complexity into subcutaneous tissue of back of athymic mouse, after implantation 4, 8, 16 weeks, sacrifice athymic mouse and take out implant body that observed tissue structure character and the differentiation situation toward tooth by HEdyeing, Masson dyeing and the immunohistochemistry dyeing of DSPP.The experimental result are: 1. Cultured hDPMCs grows good in vitro and the cells present shuttle form, triangle or polygons;Collagen I, fiberonectin and laminin show positive dyeing within the cell and its secretion matrix, which may conclude hDPMCs in vitro have the ability of the formulating and secreting matrix; hDPMCs induced by mineral solution can form mineralization tubercle which assumes the brown red by the alizaein red dyeing, this show that hDPMCs have the ability of forming calcified matrix. 2. At the 0-100 ug/ L concentration scope, bFGF and IGF-I have the action of accelerate hDPMCs proliferation, and the action of bFGF is bigger than IGF-1, two factor have the synergical action to hDPMCs proliferation, the biggest and valid concentration of bFGF is 10 ug/ L, and the biggest action concentration of IGF-1 is 100 ug/ L. At the 0-7 days time scope, the influence of bFGF on ALP activity of hDPMCs is not obvious, but with the increment of time, the action of IGF- I to the ALP activity of hDPMCs become high, and IGF-I combined with the use of bFGF can increase synergically the ALP activity of hDPMCs. 3. The hDPMCs stimulated by IGF-1 suit and IGF- I + bFGF suit, after the 7 days under the inverted microscope appear the singular plasm salience, the shape of cell likes odontoblast; The DSPP of the cell presents the positive expression by immuno- cyto- chemistry dyeing; The result of RT- PCR examination manifestated the cell expressed the hDSPPmRNA which is specificial marker of human odontoblast. The above result illuminate the hDPMCs induced appear change toward the function odontoblast. 4. The external appearance of maked PDDLLA leg froth material present ivory; The surface hole of material spread densely, holes is mutuallyconnect, the aperture is 100-300 um; there are many micropores on the wall of the foramen, the material surface is smooth;The porosity of material amounts to 90% or so;The rate of degradation of 12 weeks amount to 52%.The hDPMCs compounded the PDLLA froth leg material were cultureed in vitro after 6 hours, under inverted microscope some cell adhere to the periphery of the material and the edge of the holes, the amount of cell increase with the increment of time; Under the scanning electron microscope, after culture 1 day, hDPMCs attached to the material surface, presenting the shuttle form, triangle or polygons, and some cell crossed over the micropore surface or growed into aperture, after developping the 8 days, the cells intensively grow and get in touch with close, there is matrix formulating in parts of districts, some cells contain singular plasm prominency; The growth curve and the flow cytometry examinations of the culture hDPMCs compounded on the PDLLA material maked no difference with the hDPMCs cultured in culture bottle, these results elucidated propagation activity of hDPMCs growed at material was not affect by materials. The hDPMCs- leg material complexity was implanted into subcutaneous tissue of back of athymic mouse,a week later, the operation wound was I period cured; After 4 weeks, the physical volume of specimen took out approached to that of implantation; compared with specime of 4 weeks, the physical volume of the specimen of 8 weeks and 16 weeks became obviously lower, at 8 weeks ,it was clear that there were abundant blood vessel to go into the implant. At 8 weeks and 16 weeks, there were X - ray block project images in the implant region. The HE dyeing: After 4 weeks of implanting, material partly absorbed, the cell growed into the material back lash, with a little amount blood vessel; After 8 weeks ofimplanting, the material was further to absorb, cell component and matrix component were more, with abundant blood vessel;After 16 weeks of implanting, cell component and matrix component further increased, but still may see part of materials not to absorb. Three color dyeing: After 4 weeks of implanting, a little amount collagen was formulated in extra-cellular matrix,but the hard tissue didn't be formulated; After 8 weeks of implanting, containing abundant blood vessel, the formulated collagen is more, there was a little amount hard tissue to be formulated; After 4 weeks of implanting , there was abundant collagen to be formulated, the hard tissue increased; Not to see the dentinal tubule - like structure to be formulated. DSPP's immuno- histo- chemistry dyeing: After 4 weeks of implanting ,Part of cell and formed matrix present the positive dyeing; After 8 weeks of implanting, the matrix and cell compo- nents increased, the cell and matrix of positive dyeing increased, the positive dyeing was more thick; After 16 weeks of implanting, the cell and matrix component of the male dyeing further increased, the express was stronger.The experimental conclusion are: 1.Through the mechanical isolation and sticking wall method, we can acquire hDPMCs of the higher purity; The hDPMCs cultured in vitro have the ability of formulating extracellular matrix and mileralization and have potential is as seed cell of tooth tissue engineering to carry on to research. 2, within the certain scope of concentration and action times, the growth factor IGF- I, bFGF have importantly accommodation action to the growth and differentiation of hDPMCs, this results have the certain leading meaning to the research of the seed cell of tissue engineering tooth. 3. the IGF- I can stimulate the hDPMCs to differentiate toward the function odontoblast -like cell, the bFGF has synergy to IGF-1; The IGF-1 promote possibily hDPMCs to differentiate toward odontoblast, this laid the theories basal for open out the molecule mechanism of tooth germ development and contribute to the perfect the construction theories of tissue engineering tooth. 4. The PDLLA froth leg material of this experiment has better physical and chemical fperformance; The biological compatibility of material and the hDPMCs is better; When the compound body of hDPMCs and materials grew in nude mouse and be able to the in vivo good, hDPMCs took the differentiation toward odontoblast-like cell andformulated dentin - like structure.Based on these research, we provided the related experiment method and the technique routes for differentiation mechanism of odontoblast and the reconstruction of pulp- dentin complexity, and established theoretical groundwork for the tissue engineering pulp- dentin complexity.