Construction and experimental research of atrophic bone nonunion organoid on chip based on vascularization mechanism / 中华骨科杂志

ABSTRACT

Objective:To design and construct a bone nonunion organoid on chip and explore the mechanism of aseptic bone nonunion.Methods:First a semi-open microfluidic chip was designed, on which human bone marrow mesenchymal stromal cells (BMSC), human fetal lung fibroblast 1, (HFL1) and human umbilical vein endothelial cells (HUVEC) were co-cultured, and a three-dimensional organ on chip system was established. Different proportions of HFL1 and HUVEC were co-cultured with BMSC, which were divided into the control group (HFL1∶HUVEC=1∶1), the fibrosis group (HFL1∶HUVEC=3∶1) and the vascularization group (HFL1∶HUVEC=1∶3). The osteogenic differentiation of BMSC was observed by alkaline phosphatase (ALP) and Alizarin red staining. The transcription level of osteogenic marker genes SP7, RUNX2, ALPL, and BGLAP, and vascularization related genes KDR and VWF were analyzed by qPCR. The expression levels of RUNX2 and ALP were determined by Western Blot. Results:In the co-culture system of BMSCs, HFL1, and HUVECs, BMSCs exhibited normal growth and apparent biomineralization behavior. Endothelial cells were capable of forming structured vascular networks, confirming the successful establishment of the system. Compared to the baseline group, the fibrotic group showed no significant decrease in BMSC osteogenic differentiation. The relative expression levels of the mineralization marker genes ALPL and BGLAP were 0.55±0.19 ( P<0.001) and 0.42±0.27 ( P<0.001), respectively. Vascularization genes KDR and VWF were downregulated, with relative expression levels of 0.49±0.17 ( P<0.001) and 0.49±0.21 ( P<0.001). In contrast, in the vascularized group, BMSC osteogenic differentiation genes SP7, RUNX2, ALPL, and BGLAP were upregulated, with relative expression levels of 2.91±0.52 ( P<0.001), 3.83±1.87 ( P<0.001), 3.22±1.29 ( P<0.001), and 5.21±1.46 ( P<0.001), respectively. Vascularization genes KDR and VWF were also upregulated, with relative expressions of 8.24±2.84 ( P<0.001) and 5.32±1.67 ( P<0.001). Western blot results indicated increased expression of RUNX2 and ALP in the vascularized group and decreased expression in the fibrotic group. Conclusion:The bone nonunion organoid on chip could partially simulate the local microenvironment of bone nonunion. Fibrosis may lead to a significant decrease in bone formation ability and vascularization level, which might be an important reason for the occurrence of aseptic bone nonunion.