Construction Of Tissue Engineering Scaffolds With Alginate Microchannels Based On Coaxial Bioprinting

Part 1 Two-step construction of alginate tissue engineering scaffoldsResearch Background:Two-dimensional（2D）research platform is often used for drug screening in vitro,and with the difference of 2D and 3D spatial structure in cell-cell and cell matrix,that often leads to the difference of intracellular signal molecule expression and distribution,and finally result in the significant difference of cell morphology and function.Therefore,in addition to using in regenerative medicine,the products of 3D tissue engineering which is constructed by tissue engineering technology,also play an important role in new drug screening as a highly biomimetic drug screening platform.Bioprinting is a kind of biological additive technology,which can print different cells and biomaterials to precise spatial locations by computer-aided design technology,and finally realize the automatic construction of highly biomimetic active 3D tissue through layer-by-layer printing.The construction of tissue engineering products by bioprinting has achieved rapid development in the past decade.As a special bioprinting method,coaxial printing technology has an important application prospect in the fields of regenerative medicine and new drug research and development due to its convenient printing of diverse sheath/core structure tissue engineering scaffolds.Objective:At present,researches of coaxial printing are all through a"one-step"method to construct tissue engineering scaffolds,which can simultaneously complete coaxial printing scaffolds and three-dimensional tissue construction.In this study,we propose a"two step"method to construct alginate tissue engineering scaffolds.The first step is to print a three-dimensional alginate hollow scaffold by coaxial printing,and the second step is to perfuse cells and hydrogel matrix,which can stabilize and facilitate the construction of various matrix tissue engineering scaffold.This study intends to verify the feasibility of infusing different types of hydrogel matrices with scaffolds constructed by a"two-step method".The stability and wide applicability of the"two step method"are further confirmed by detecting the effects of crosslinking agent concentration,hydrogel type,cell density,mixing ratio and number of scaffolds layers on cell viability and function.Methods:（1）With the help of a coaxial nozzle and a 3D bioprinting system,a multilayer alginate holder with a micro-channel structure was printed to verify the effect of material concentration on the printability of the scaffolds.（2）Print the alginate scaffold with a 4-layer structure at the optimal printing density and determine the mechanical properties of the scaffold.（3）Gelma solution with concentration of 30%was infused into different layers of alginate hollow scaffolds,performed light crosslinking under 312 nm ultraviolet light in the presence or absence of medium,and observe the effects of the presence of the medium and the number of scaffold layers on GelMA light crosslinking.（4）Observe the effects of the concentration of the crosslinking agent,cell density,hydrogel type,and cell to hydrogel mixing ratio on cell viability by dead-and-live staining.（6）Long-term culture of cell hydrogel mixture poured into different layers of scaffold:The cell viability in the scaffold was detected on the 1st,3rd,6th,and 9th days by dead-and-live staining,Alarma Blue kit was used to detect cell proliferation,determination of albumin secretion by ELISA Kit,modified Jung method was used to detect the amount of urea synthesis.Results:（1）When the concentration of sodium alginate was 3%-4%and the concentration of CaCl₂ was 2%-4%,coaxial bioprinting had better printability,the printed alginate with multi-layer scaffolds had a uniform structure,complete and continuous internal channels,and can support the perfusion of gas and liquid.（2）The mechanical properties test results showed that the 4-layer alginate scaffold printed with 3%alginate solution had high compression modulus.（3）The light crosslinking reaction of GelMA hydrogel in the presence of or without media was observed,and the crosslinking time was affected by the number of scaffold layers.The crosslinking time of the first layer was about 1.5 min,that of the second layer was about 2.5 min and that of the fourth layer was about 6.5 min,that of the eighth layer was about 17min and that of the tenth layer was about 20 min,and that of the 11th layer was over30 min.（4）The cell survival rate of the uncrosslinked group was 95.73%±2.78%,the cell activity in the group with the crosslinker concentration of 1%,2%,and 3%was 95.67%±1.17%,95.07%±2.38%,and 89.4%±0.67%,respectively,and there was no significant difference（P>0.05）.（5）The results of life and death staining showed that the cell density,the type of hydrogel,and the mixing ratio of cells to hydrogel in the scaffold had no effect on cell viability,and the cell viability in each group was>90%,there was no significant difference（P>0.05）.（6）The results of life and death staining showed that the cell viability of the 4-layer and 8-layer scaffolds on days 1,3,6,and 9 were all>90%,and the results were not significantly different（P>0.05）.Alarma Blue test results show that the proliferation rate of cells in the8-layer scaffold on days 1,3,6,and 9 was about twice that of the 4-layer scaffold.The results of ELISA and QuantiChromTM Urea showed that the total amount of albumin and urea secreted by the 8-layer scaffold on days 1,3,6,and 9 was about twice that of the 4-layer scaffold.It proved that the number of scaffold layers has no effect on cell activity and secretion function.Conclusion:Based on the"two-step"coaxial bioprinting method,three-dimensional alginate multi-layer tissue engineering scaffolds can be effectively prepared,this method supports different kinds of hydrogels,stable growth and proliferation of cells in scaffolds,it shows that the construction method has good feasibility,stability and applicability.Part 2 Coaxial printing perfusable dual-channel alginate tissue engineering scaffoldObjective:Construction of alginate tissue engineering scaffold with built-in dual channels by coaxial technology,establish a perfusion culture method,and verify the influence of perfusion culture on the activity and function of cells in the scaffold.Methods:（1）Used a special coaxial nozzle to print a perfusable dual-channel alginate scaffolds to verify the effect of printing parameters on the diameter of the dual-channel tube and the wall thickness between the two tubes.（2）The HepG2 cells with a density of 2×10⁶ cells/ml and a concentration of 1%sodium alginate solution were mixed according to 1:1 and then infused into one side of the double channel tube,putted it in 1%CaCl₂ solution,after it is crosslinked,connect the other side channel of the double channel pipe to the perfusion device for perfusion culture,the cell viability under perfusion culture and routine culture was measured on day 1,3,5and 7 using dead and alive staining kits;CCK-8 kit was used to detect the proliferation ability of the cells;under the condition of perfusion culture,the cell culture medium of 1,3,5,7 days was collected,the urea synthesis was measured by modified Jung method,and the albumin secretion was measured by ELISA.Results:（1）The results of optical microscopy showed that the internal and external diameters of the scaffolds increased with the increase of the outflow velocity of CaCl₂ solution and the extrusion velocity of sodium alginate solution,the difference was statistically significant（P<0.05）.The concentration of calcium chloride solution and sodium alginate solution had no regular effect on the pipe diameter;the wall thickness between the two hollow tubes was related to the concentration of CaCl₂ and sodium alginate solution,the greater the extrusion rate of the sodium alginate solution,the thicker the wall between the two hollow tubes,the greater the outflow of calcium chloride solution,and the thinner the wall between the two hollow tubes,the difference was statistically significant（P<0.05）.（2）Compared with the conventional culture,the cell activity of perfusion culture was similar to that of conventional culture on the first day,the activity of the 3rd,5th and 7th cells was significantly higher than that of the conventional culture（P<0.05）.CCK-8 test results showed that under the condition of perfusion culture,the proliferation of cells increased on day 1,3 and 5,and decreased on day 7.The results of ELISA showed that under the condition of perfusion culture,the secretion of albumin increased on the 1st,3rd and 5th day,the highest on the 5th day and decreased on the 7th day.QuantiChromTM Urea test results showed that the trend of urea secretion was the same as that of albumin.Conclusion:Constructed perfusable dual-channel alginate scaffold by coaxial bioprinting technology,and a perfusion culture method was established,which significantly improved cell activity and maintained cell secretion function.It has good feasibility in terms of activity and function.