The Preparation And Preliminary Characteristics Detection Of Cell-laden Alginate-gelatin-bioglass Bioink

Tissue and organ destruction and loss caused by trauma or genetic defects has become a serious problem affecting human health.Currently,the main solution is transplantation-treatment.Because of the limited source of donors,artificial implant is considered to be a means to alleviate or solve tissue or organ problems.Using 3D bio-printing technology to construct artificial grafts is a common method in tissue engineering research.3D bioprinting can simulate extracellular matrix,even replace extracellular matrix,and provide a highly simulated cellular microenvironment for cells in vitro.Bio-ink is the key to promote the development of cell-laden 3D bioprinting technology.The ideal bio-ink should have good physical and chemical characteristics and biocompatibility.Natural polymers are listed as the first choice for bio-ink because of their clear structure and convenient materials.Sodium alginate and gelatin are two kinds of natural polymers which widely exist in nature.Alginate-gelatin composite bio-ink(Alginate-Gelatin bioink,Alg-Gel)has good printability,pore structure and biological properties,but it can not provide high mechanical hardness,as a result it becomes a hot research to add synthetic materials to improve Alg-Gel bioink mechanical properties.Silica-based nano-bioglass(Bioglass,BG)is a kind of synthetic material with hard structure,which is expected to improve the mechanical properties of Alg-Gel bioink.Objective:To improve the physical and chemical properties by adding BG into Alg-Gel bioink,and to study the biological characteristics of Alg-Gel-BG bio-ink.Methods:1.Preparation of BG and composite hydrogels:the bioglass nanoparticles was prepared by calcium hydroxide saturated solution and silica nanoparticles;Alg-Gel-BG biohydrogel was prepared by adding BG into Alg-Gel hydrogel.2.Physical and chemical characteristics test:The rheology,shear rate,young’s Modulus,water content,porosity and porosity rate of Alg-Gel-BG Hydrogel were tested separately.3.Cell extraction and hydrogel cell viability assay:dermal fibroblasts were isolated and co-cultured with hydrogel,the viability and inhibitive ability were tested by CCK8 assay.4.Construction of cell-loaded 3D bioprinting scaffold:dermal fibroblasts were fused with bio-ink to print 3D scaffold.5.Detection of cell viability,adhesion and extension of biological scaffolds:Ki67 immunofluorescence was used to detect cell proliferation on the 3rd day;Cell adhesion and elongation were detected by scanning electron microscopy;Cell viability and extension were detected by immunofluorescence assay with Paxilin andβ-actin anti markers.6.Statistical analysis:The data of this experiment were all metrological data,expressed as X±SD,and analyzed by SPSS 19.0 software.The t-test was used for double-group data,One-way analysis of variance(ANOVA)was used for more than three groups of independent samples,and repeated measures analysis of variance(ANOVA)was used for repeated measures data,P<0.05 was considered to be statistically significant.Results:1.The bioglass nanoparticles were well dispersed in water evenly,and the best combined solid content of BG and Alg-Gel hydrogel were 0.5wt%and 1.0wt%.2.Physical and chemical characteristics test results:the gelation time of Alg-Gel-BG hydrogel is less than 150 seconds at room temperature,and the viscosity of Alg-Gel-BG hydrogel was decreased with the increase of shear rate;the mechanical hardness of Alg-Gel-BG hydrogel was higher than that of Alg-Gel hydrogel,which is a porous structure as Alg-Gel-BG hydrogel.3.Dermal fibroblasts were isolated successfully.As the results of co-culturing of fibroblast and hydrogel suggested,the cellular viability of Alg-Gel-BG hydrogels were better than that of Alg-Gel hydrogel.4.The extrusion filament process of Alg-Gel-BG bioink on the bio-printer was continuously,and the 3D printing constructions of cell-laden Alg-Gel-BG bioink were complete with clear interval space and continuously solid construct.5.The cells could be adhered to the Alg-Gel-BG 3D bio-printing scaffolds and retain high viability,proliferation and extension.Conclusion:Alg-Gel-BG hydrogels were prepared by adding nano-bioglass into alginate-gelatin composite hydrogels they all had good physical and chemical characteristics,allowing cell to adhere,survival,proliferate and extend in their 3D scaffolds,it may be a good idea to treat the Alg-Gel-BG bioink as second choice for 3D bioprinting.It is expected to provide more laboratory or clinical printing models and further promote the research progress of artificial organs.