| Background: Tissue engineering provides a good method for the study of skin substitutes,but the current bottleneck is hard to solve.This study focused on the construction of artificial skin by bio-3D printing,developed a bio-based 3D printer that can be used for artificial skin construction,and explored the construction of artificial skin by bio-3D printing in vitro.Part I: The first part: bio-type 3D printer research and development and testingObjective: To develop and test a bio-based 3D printer that can be used to make bio-3D printed artificial skin.Methods: In order to realize the construction of artificial skin in vitro through a bio-based 3D printer,the research group proposed the design requirements of the bio-type 3D printer equipment required by the research project and handed over the equipment manufacturing to the equipment company.After the biotech 3D printer PrototypeSK00 was manufactured,3D models of two different parameters(Cylinder001.stl and Cylinder002.stl)were designed by 3D Studio Max software and parsed into goc files by RepetierHost software.The prototypes were configured with 1%,2.5% and 5% Based on two different parameters of the model and three different print components tested five different inner diameter of the print head(0.09 mm,0.16 mm,0.25 mm,0.41 mm and 1.00mm)operation.Fibroblasts and epidermal cells were digested to form a cell density of 1 × 108 / ml DMEM suspension,the two kinds of cells through different inner diameter of the print head direct printing planted in 12-well plates,after detection of printing operation 0h,6h,24 h And 48 h cell survival rate,the control group is directly through the micropipette cell implantation in culture wells.In addition,in order to test the stability of the bio-based 3D printer PrototypeSK00 in the construction of artificial organs,a three-dimensional model of the nasal simulation was designed and tested in vitro using 2.5% sodium alginate and 0.41 mm printheads.The control was the same three-dimensional model in the industrial Polylactic acid artificial nasal model constructed by 3D printer.Results: The project team designed and completed the fabrication of a biometric 3D printer,PrototypeSK00.The run-in process found plugging on the basis of a 0.09-mm print of high-sodium alginate solution.When printing sodium alginate solution with lower concentration,the model with the larger total volume is prone to evaporate after the printing,resulting in the failure of the pre-built model to dry and collapse.Fibroblasts and epidermal cells after printing operation,0h-48 h cell survival rate of the lowest 95%,up to 100%,no significant difference with the control group.The artificial nose of the Prototype SK00,a bio-based 3D printer,is close to the artificial nose model built with industrial 3D printers.Conclusion: The research team developed a relatively stable biological type 3D printer PrototypeSK00,its operation process no obvious error occurs,after its printing operation cell survival rate and micropipettor no difference,through the printing can be stable operation In vitro construction of advanced artificial structure.Part II: R & D and Testing of Skin "Bio-ink" Applied to Bio-type 3D PrintersObjective: To develop a "bio-ink" as a scaffold that can be used in biological 3D printers to construct artificial skin in vitro.Methods: The recombinant collagen and alginate solution were mixed and sterilized to form "bio-ink".The reference substance was alginate solution without collagen component.Some samples were lyophilized to form a solid state,and the physical characterization of the material was observed by scanning electron microscopy.The hydrodynamics of the sample was also measured on a fluid rotation rheometer.In order to test the cytotoxicity of "bio-ink",the experiment was carried out by passing on the cultured fibroblasts and epidermal cells.The effect of this "bio-ink" on repairing wounds was tested by the full-thickness skin defect model of bal / bc nude mice and SD rats.The pathological changes were observed on the 1st,2nd,3rd and 4th weeks,and the wound healing,vascularization,Situation and epidermal crawling situation.Results: A highly bioactive collagen "bio-ink" with uniform pore structure was constructed by recombinant human collagen without obvious toxicity to epidermal cells and fibroblasts.Nude mice wound healing experiments confirmed that the collagen-containing "bio-ink" can help promote vascularization of the skin,promote the recovery of the dermal layer thickness and increase the collagen content of the wound.Conclusion: The research contents of this section are: "Bio-ink" which is similar to the skin's extracellular matrix by using recombinant human type III collagen as bio-ink material used in the subject,and to test its biological toxicity and Wound healing effect,which itself has to accelerate wound healing and improve the quality of wound healing,in order to build biological 3D printed artificial skin preparation.Part III: the preliminary construction and testing of artificial skin based on bio-type 3D printerObjective: Based on the self-developed biotype 3D printer PrototypeSK00 and the developed collagen "bio-ink",combined with fibroblasts and epidermal cells,a preliminary study of the biomimetic 3D biopsy artificial skin was conducted by means of biological 3D printing The preliminary biological evaluation.Methods: The software of 3D Studio Max program(Autodesk Corporation,USA)was used as the model to establish the multi-level three-dimensional model data of dermis and epidermis.Fibroblasts and epidermal cells were mixed into "bio-ink" to perform in vitro 3D printing simulating human skin structure,and immediately frozen sections were combined with HE staining to observe the microstructure.Fibroblasts stably expressing EGFP protein were constructed by transfection of EGFP gene sequences in fibroblasts and screened with puromycin.The multi-layer structure was constructed by using this cell system for in vitro 3D printing and observed under a fluorescence microscope Cell status after 7 days of in vitro culture.Results: Based on bio-based 3D printer and "bio-ink" results,in vitro bio-3D printing of artificial skin was completed.HE staining after frozen sections showed that the "dermis" and "epidermis" were clearly distinguished.Multilayered structures by 3D printing with EGFP-stably expressing fibroblasts after 7 days of in vitro culture demonstrated that fibroblasts survived "bio-ink" and that underlying cells adherently proliferated.Conclusion: By means of biological 3D printing,collagen "bio-ink" is used as a scaffold,and fibroblasts and epidermal cells are seed cells,which can simulate the skin structure in vitro to construct artificial skin. |
PDF Full Text Request