A 3D Bone Tissue Chip Based On Microfluidic Technology

Objective: Currently,the gold standard for preclinical drug screening and innovative treatment concept validation studies in bone tissue engineering is the use of animal models.However,there are certain differences in physiological and metabolic processes between different species.In addition,traditional 2D cell culture cannot support the tissue-specific and differentiation functions of multiple cell types,nor can it accurately predict the in vivo tissue function and drug activity,and lacks the interaction between cells and extracellular matrix.Compared to cells grown in 2D monolayer,3D bone tissue cell spheres have better biological functions,higher cell density,stronger resistance to drugs,and more accurate responses to drug concentrations.Furthermore,it can better simulate the intercellular metabolic interaction in the three-dimensional space of in vitro cells.Organ chips are miniature cultivation devices that simulate the main structure and functional characteristics of human organs based on microfluidic technology,which is the core of the organ chips.In view of the existing in vitro models for bone tissue engineering that cannot well simulate the in vivo drug response,are time-consuming,expensive,and have low success rates,this study based on microfluidic technology,designs and builds a highthroughput 3D vascularized bone tissue organ chip for drug screening and material evaluation.Methods: The top layer of the chip was made using a traditional photolithography process to create a silicon wafer mold.The bottom layer was designed as a U-shaped groove structure,made using a high-precision 3D printing process,and smoothed to provide a suitable concave structure for cell aggregation growth.To build a threedimensional bone tissue chip,human bone marrow stromal cells and human umbilical vein endothelial cells were selected for three-dimensional co-culture and osteogenic induction in vitro.The activity of the three-dimensional bone tissue cells was then detected,and the cell sphere shape was characterized,as well as specific bone and blood vessel endothelial functions.Additionally,the chip platform was used to evaluate the effects of mineralized collagen(MC)on bone formation and angiogenesis.Results: The results of the experiment confirmed that cells can spontaneously gather and grow in the grooved chip to form dense three-dimensional microsphere organizations,which can more realistically simulate the microenvironment of bone tissue in the human body.The cell balls showed good cell activity and proliferative ability when subjected to activity detection.The three-dimensional cell ball morphology was characterized and observed that cells were evenly distributed from the center to the edge,the cell ball had a dense cell skeleton structure,and the endothelial cells were evenly distributed throughout the cell ball and maintained their endothelial phenotype.By testing the osteogenic-specific function and vasculogenic-specific function of the cell ball,the results showed that the cells on the ball were induced to differentiate into bone,and showed the formation of a blood vessel precapillary network.Testing the known mineralized collagen that has a good promoting effect on bone formation confirmed the results and showed that the three-dimensional bone tissue organ chip can be used in drug screening,material evaluation,and disease modeling in bone tissue engineering.Conclusion: In this study,based on microfluidic technology and the physiological structure of human bone tissue,we designed and fabricated a high-throughput threedimensional vasculogenic bone tissue organ chip and applied the chip to evaluate the potential of mineralized collagen to promote bone formation and vasculogenesis.Our research results indicate that the high-throughput three-dimensional bone tissue organ chip provides a new platform for drug screening and development in bone tissue engineering,in response to the current limitations of in vitro models,which cannot well simulate the drug response in the body,as well as the long time,high cost,and low success rate.