Development Of An Extrusion Bioprinter And Evaluation Of An Engineered Cartilage With Biomimetic Chondrocyte Density Gradient In Vitro

BackgroundThe cell,the element of life,has become a printing material to fabricate tissues and organs when the bioprinting technology birthed out as a landmark of the integration of the 3D printing and the life science.Different from conventional 3D printing technologies,the bioprinting takes more consideration on cells' biological behavior than the product's manufacturing precision and mechanical strength.The whole system of the bioprinting set up with cells as core,including developing a printing platform,designing a printing plan,and evaluating the bio-printed construct,et al.To date,the bioprinting technology is still in its nascent stage due to complicated and various characters of the life science.Efforts are needed to achieve the goal of fabricating functional tissues and organs.Developing a bioprinter is an essential prerequisite of achieving the bioprinting,and also one of challenges needed to be conquered.It represents a high level comprehensive application of the cell biology,biomaterials science,automatic control,and information science.In a sense,having the ability of developing a bioprinter means mastering the core of the bioprinting.There are many setup forms of the bioprinter on the conditions of different printing materials.However,none of them is perfect.In the present,some commercial bioprinters could be offered by a few developed countries.Having the advantages of high level system integration and easy manipulation,the commercial bioprinter also has the obvious deficiencies including limited printing materials,fixed parameter setting,and et al.In addition,it is not feasible to upgrade software and hardware systems due to the patent protection.As a result,the commercial bioprinter has not been used widely.Articular cartilage consists of the chondrocyte and extracellular matrix(ECM).It is aneural,avascular,and alymphatic and has limited self-repair ability.Once cartilage defects are caused,it inevitably leads to progressive joint damage.Unfortunately,none of the current treatments for cartilage defects has a long time satisfactory outcome.Cartilage tissue engineering is a promising approach to repair and regenerate cartilage tissue with a combination of manufacturing technology and life science.Engineering zonal cartilage is one of the current research hotspots,which aims to mimic zonal properties of the articular cartilage in structure and function.The cell density gradient is one of zonal properties in the mammal's articular cartilage.Few studies have focused on engineering a cartilage with biomimetic cell density gradient.This study intended to develop a hydrogel extrusion bioprinter,to bioprint an engineered cartilage with biomimetic chondrocyte density gradient,to investigate the biological effects of cell density gradient in in vitro culture,and as a result,to achieve the aim of mastering the bioprinting technology.MethodsIn this study,a bioprinter was developed by our lab based on open resource systems,which consists of the Prusa i3 three-axis motion system and a piston-driven microscale depositional equipment.Softwares used in this study included Solidworks,Slic3 r,and Repetier-Host.A 3 m L syringe was used as the bioink cartridge.A 25-gauge needle used as the nozzle.10% Collagen type II derived from swine knees in 0.15 M Hydrochloric acid solution was used as the bioink.The 3D solid model was created in Solidworks and then transected into a series of 2D contour profile in Slic3 r after the printing parameters having been set including layer thickness,nozzle moving path,printing speed.The bioprinter was controlled by a Gcode file.A printed collagen type II hydrogel construct crosslinked at 37? in a humidified incubator.This study intended to construct an engineered cartilage with biomimetic chondrocyte density gradient through bioprinting technology,Based on the total cell density within the articular cartilage of the human medial femoral condyle(1×107 cells/m L)and the ratio of chondrocyte densities in the superficial zone to the transitional zone to the deep zone of normal adult human articular cartilage(3:2:1),an engineered cartilage was fabricated using chondrocyte-laden collagen type II hydrogel as bioink.Engineered cartilage were cultured in vitro and harvested at 0,1,2,3 weeks for cell viability test,RT-PCR,biochemical assays,and histological analysis.ResultsA hydrogel extrusion bioprinter was successfully developed.The high cell viability in printed constructs was remained by optimizing the printing parameters.During the 3-week in vitro culture,COL1A1 expression remained low,and it was down-regulated throughout the culture period.The expression levels of both COL2A1 and ACAN were significantly increased(p<0.05).A slight decrease in the total cell number was observed in all groups during culture,but the difference was not statistically significant.During the first week of culture,the GAG content had a positively correlation with the total cell density.Group A had the highest GAG content,and Group C had the lowest.During the second and third week,only Group C had a lower GAG content compared to Group A or B(p<0.05).The differences between Group A and B were not significant.To assess the average single-cell GAG production,the total GAG content was normalized to the cell number.In the first week,there were no significant differences among the three groups.In the last two weeks,the average single-cell GAG production in Group A was the lowest among the three groups.Group B had the highest average single-cell GAG production.Significant differences between the construct with the cell density gradient in Group B and the constructs in Group A and C were observed,although there was no significant difference within Group B.At 3 weeks,a significant difference was observed in the constructs with the homogeneous cell distribution between Group B and Group C.The results of Alcian blue staining for GAG and immunohistochemical analysis of collagen types I and II and PRG4 showed that the positively stained cells were brown,the negatively stained cells exhibited blue nuclei,and GAG in the matrix was also stained blue.Nearly all of the chondrocytes in the constructs stained positively for collagen type II,many stained positively for PRG4,and a few stained positively for collagen type I.In the constructs with the cell density gradient,the collagen type II,PRG4 and GAG contents were concentrated in the superficial zone and decreased with depth.ConclusionOur results suggested that it is feasible to develop a hydrogel extrusion bioprinter based on open resource systems,that an engineered cartilage with biomimetic cell density gradient could be constructed through the bioprinting technology,the synergic biological effects could be achieved by optimizing the total cell density and cell distribution pattern,bioprinting collagen type?hydrogel constructs with biomimetic chondrocyte density is a new strategy to construct a zonal engineered cartilage.