| The target of regeneration of tissue or organ is the complete reconstruction of structure and function of failed tissue or organ. Dentistry researchers eagerly welcome three-dimensional cell printing methodin tissue engineering of whole tooth, because it may represent an ideal organ of alternative medicine research. Human teeth are replaced only once during life, andthe enamel cannot regenerate. Therefore, when the teeth or enamel lost, the artificial reconstruction would be needed. The purpose of researches on dental tissue engineering and stem cell biology is to propose the strategy and method for determining the replacement of lost or damaged teeth. The special tissue derived from stem cells, such as dentin and bone, has clinical application, but the method of tooth tissue engineering is still in research.A basic requirement of 3D digital engineering of complex and heterogenous architechture of functional tissues and organs is to make clear and comprehensive understanding of the structure and organization of their components. Medical imaging technology is an indispensable tool for tissue engineering to provide information on three-dimensional structure and function at the cell, tissue, organ until the organism level. These techniques include computed tomography and magnetic resonance imaging, Computer aided design and computer aided manufacturing tools and mathematical modelingsare used to collect and digitalize the complex structure and the fault information of the target tissue. The completed tissue or organ model is used for digitally controlled biological printing design and manufacturing system. System software can complete the model of three-dimensional reconstruction and reslice the 3D model to two-dimensional images, makes the 3D model being divided into two-dimensional slices, and then import them to the biological printing system. Data of anatomy and structure information contained in two-dimensional horizontal slices can provide the bio-printer printing instructions of different cells and extracellular matrix layer by layer. We require a 3D inkjet printer system for tissue engineering based on biological cells. In the first part of this study we used a variety of methods to construct three dimensional digital tooth germ models as three-dimensional inkjet printing template.In the first experimentof part one, as representatives of different developmental stages, a neonatal dog, a dog of 4 weeks and a dog of 5 months were used for the study of tooth germ development status by using the Micro CT and CBCT. The results showed that the deciduous crowns were mineralized in neonatal dog, and there was no obvious development of permanent teeth germs. In 5 months old dog, being in the start stage in the replacement of primary teeth, the permanent tooth crown had been mineralized, and most of teeth were in the replacement period. In 4 weeks old dog, the deciduous teeth were at the start of eruption period, the fourth premolar and the first molar tooth germ were respectively 967.4 mm3 and 46.6 mm3, and suitable for the subsequent study on 3D cell printing of tooth germ in non-mineralized bell stage as 3D digital template.This was the first study to build the three-dimensional digital tooth germ model at 20μm super fine resolution. The resolution of the model was at the cellular level, which was to say one pixel would be corresponding to one cell. This would meet the basic demand of single cell resolution inkjet printing. The 3D tooth germ template could be transformed and identified to a variety of 3D digital file format, could be resliced at any angle and layer thickness. And two-dimensional print template data could be generated at different layer thickness and resolution, so as to meet the need of three-dimensional bio-printing system.The 3D digital template, being constructed with Micro CT data, could not achieve fine spatial positioning of various types of cells in tooth germ and cell density distribution, and the extracellular matrix and the percentage of cells also could not been analyzed, the capillary network structure also cannot been distinguished. This required constructing the ultra precision 3D digital tooth germ template by the further use of tissue pathology, to meet the accurate localization and density of multicells and extracellular matrix.In experiment two of part one, we chose the first molar germ and fourth permanent premolar germ of 4 weeks old dog as the study object of 3D histological reconstruction. HE staining was used to stain the continuous 730 sections of tooth germ, 684 of which were valid slices. Pannoramic SCAN digital section scanning system was used to sequentially scan the valid slices, data were obtained at resolution of 0.52μm/pixel. Tissue biopsies were conducted and aligned by three-dimensional Micro CT reconstruction data of experimental one, and the aligned and registered slices were imported into Pannoramic Viewer software, then 3D reconstruction of tooth slice data was carried out and the three-dimensional digital model of early development stage of tooth germ with the micron-resolution was constructed. The model can be transformed into a variety of formats by three-dimensional software, and suitable for different needs.In experiment three of part one, Pannoramic Viewer and Pannoramic Histo Quant software were used for computer graphics analysis of 3D digital cytology model, in research on the spatial distribution and positioning of various cells, and the extracellular matrix and the microvascular network. And then, we can provide solutions for the reasonable design for cell-contenting bio-ink composition, and printing template that can be identified by three-dimensional printing software. The results showed that the cell density is about 44.74×106/ml in dental papilla region, the cell density of 16.20×106/ml in dental follicle region, and the cell density of 8.30×106/ml in stellate reticulum. The outer enamel epithelium cells were in 5-8 layers with the width of 50-60μm and cell density of 3-4×108/ml. Inner enamel epithelium cells were in 4-6 layers with the width of 40-50μm and cell density of 8-10×108/ml. The odontoblast cells were 3-5 layers with the width of 30-40μm and cell density of 8-10×108/ml. Root sheath epithelial cells density was 3-5×108/ml. The cell volume of dental follicle is 0.021 ml per ml, the volume of dental papilla cells is 0.137 ml per ml, the volume of stellate reticulum cells is 0.007 ml per ml, and the rest volume was the extracellular matrix. The vascular network was presented in dental follicle and dental papilla, while no vascular network existed in the stellate reticulum region. The vascular network density was 26.3±4.6/mm2 in dental papilla,and the distribution of vascular network was not uniformity. The 3D vascular network can be constructed by the Imaris Software to provide follow-up vascular network printing template. The distribution and density of various types of cells obtained in the model can be used for the theoretical basis of composition of bio-ink.In the second part of this study, we cultured cells associated with dental germ development, constructed bio-ink based on sodium alginate hydrogel, studied the physicochemical characteristics of printing-stable bio-ink by using droplet observation instrument and other equipments, accessed to a stable component of bio-ink. Dental papilla cells were induced by osteogenesis differentiation in vitro. The influences of printing process on the biological properties, cell differentiation, and cell proliferation activity were studied. These studies provided the necessary technical supports for further three-dimensional multi-cell printing to construct tissue engineering tooth germ.In experiment one of second part, dental papilla cell was cultured based on the early stage of dental germ acquired from the dog in the first part experiment and its biological characteristics were studied. Cell surface antigens were analyzed by flow cytometer. Results showed that hematopoietic stem cell marker expression was negative, and mesenchymal cell marker expression was positive. ALP activity of dental papilla cells after osteogenic induction was higher than that of control group in vitro; osteocalcin content was about 20 times of the control group cells. In osteogenic induction group cells, calcium nodules appeared obvious, and Calcein fluorescence quantitative analysis results showed that the fluorescence intensity was obviously higher than those of control group. Research showed that the dental papilla cells were derived from mesenchymal stem cells with osteogenic differentiation ability.In experiment two, bio-ink based on sodium alginate was prepared, and the measurements of liquid viscosity and surface tension were performed by using ARG2 rheometer and FM40 Easy Drop contact angle measurement. We prepared the bio-ink of 1% sodium alginate, and added 0.05% sodium citrate into the DMEM culture medium to antagonize calcium ion, 0.1% poloxamer 188 and fluorinated surfactant 0.01%Capstone FS-3100 or 0.05% Novec FC-4430 to lower the surface tension. The viscosity and the surface tension properties met the need of piezoelectric nozzle printing head. XAAR128 piezoelectric array printing head and droplet observation instrument were used to test the cell bio-ink printing status by optimizing the cell concentration of 1.2×107/ml. Results showed that can the print head can achieve an average one cell in each printing ink drop.In experiment three, study on the influence of jet printing process on cell viability, proliferation, differentiation and cell characteristics were performed. The dental papilla cells at a concentration of 6×106/ml cells were suspended in surfactant containing bio-ink, 5×104 cells were printed into 12-well-plate and 1ml DMEM culture medium was added, the cells were defined as the printing group; The same number cells with same concentration of cell suspended in the bio-ink without surfactant were defined as surfactant-free group; 5×104 dental papilla cells directly suspended into the 1ml culture medium were defined as control group. The results showed that the survival activity of dental papilla cells suspended in surfactant-free bio-ink was slightly higher than that of control group, dental papilla cells of inkjet printing group remains >95% cell survival relative to the control group, and the proliferation rate after 48 hours had no significant difference between the groups. Osteocalcin expression of the printed papilla cells after osteogenic differentiation and the control group had no significant difference; the results showed that, the ink-jet printing had no obvious effect on the osteogenic differentiation of dental papilla cells. This study provided further proof that the bio-ink of alginate had characteristics of stable printing, and could be ejected from the XAAR128 printing head stably, while maintaining the printing cell density and cell viability and proliferation ability. This research made it possible that the commercial printing head could print many kinds of cells with high flux to perfectly construct of tissues and organs.In the further study, we need to focus on the research of 3D bio printer that could inkjet print a variety of cells and materials simultaneously, which is the basic demand for manufacturing multilayer cell structure and larger organs. |
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