ABSTRACT
Objective:To construct a double-layer bone-on-a-chip containing bone matrix, with which the process of osteoblast and osteoclast differentiation in vitro is stimulated, aiming to provide a new platform for the development of osteoporosis medications. Methods:Software WorkSoild was used to design the double-layer and double-channel bone-on-a-chip and the template was fabricated by photolithography. With polydimethylsiloxane (PDMS) as the raw material, the main body of the chip was prepared by mold fabrication. The inlets and outlets of the four channels of the culture room were separated with bovine cortex bones and sealed with liquid storage columns. In the chip verification experiment, chips were divided into osteogenic and osteoclastic induction groups and osteogenic and osteoclastic control groups. In the osteogenic and osteoclastic induction groups, precursor cells of mouse embryonic osteoblast, MC3T3-E1 and mouse macrophage RAW264.7 were inoculated on the chip separately. Osteogenic induction lasted 14 days and osteoclastic induction 7 days. MC3T3-E1 cells and RAW264.7 cells were not induced in the osteogenic and osteoclastic control groups. The following indicators were observed: (1) Appearance and sealing performance of the chip: After the chip was prepared, photos were taken to observe its appearance and sealing tests were conducted to observe its sealing performance. (2) Biocompatibility: At 3 days after MC3T3-E1 cells were inoculated onto the chip and cultured and at 1, 3 and 5 days after RAW264.7 cells were inoculated onto the chip and cultured, the cell survival was observed with calcein acetoxymethyl ester/propidium iodide (AM/PI) staining and Cell Counting Kit 8 (CCK-8). (3) Osteogenic differentiation: Alkaline phosphatase (ALP) staining and alizarin red staining were performed on the cells in the osteogenic induction group to observe the osteogenic induction. RNA was collected from the osteogenic induction group and the osteogenic control group, the expression of osteoblast marker Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and type I collagen (COL1A1) was detected by real-time florescent quantitative PCR (qPCR), and the differentiation degree and osteogenic ability of osteoblasts were observed. (4) Osteoclast differentiation: tartrate-resistant acid phosphatase (TRAP) staining was performed on cells in the osteoclastic induction group to observe osteoclast differentiation. RNA was extracted from the osteoclastic induction group and the osteoclastic control group for qPCR of osteoclast differentiation-related genes, and the expression levels of the osteoclast marker gene TRAP, cathepsin K (CTSK) and dendritic cell specific transmembrane protein (DC-STAMP) were detected.Results:The double-layer bone-on-a-chip containing bone matrix was 3 cm×3 cm in size and transparent as a whole. The structure of the system on the chip system was compact and had no seepage. It was shown by calcein AM/PI staining that at 3 days after MC3T3-E1 cells and RAW264.7 cells were cultured, very few red fluorescent dead cells were found. CCK-8 test showed that within 5 days after being cultured, the cell viability was all above 90%, indicating that the biocompatibility of the chip was good and the cells could survive and proliferate normally. The results of ALP and alizarin red staining showed that MC3T3-E1 cells successfully differentiated into osteoblasts and produced calcified nodules in the osteogenic induction group at 14 days after the induction. The qPCR results showed that the relative expression level of RUNX2 in MC3T3-E1 cells in the osteogenic induction group was 4.98±0.74, which was significantly higher than that of the control group (0.99±0.03) ( P<0.01). The relative expression level of OCN in MC3T3-E1 cells was 7.98±0.76, which was significantly higher than that of the control group (1.00±0.06) ( P<0.01). The relative expression level of COL1A1 in MC3T3-E1 cells was 7.07±0.56, which was significantly higher than that of the control group (0.97±0.03) ( P<0.01). The TRAP staining results showed that the RAW264.7 cells in the osteoclastic induction group differentiated to giant multinucleated osteoclasts, and TRAP protein was expressed in large quantity in the osteoclasts. The results of qPCR showed that the relative expression level of TRAP in RAW264.7 cells in the osteoclastic induction group was 3.35±0.37, which was significantly higher than that of the control group (1.01±0.06) ( P<0.01). The relative expression level of CTSK in RAW264.7 cells was 3.46±0.79, which was significantly higher than that of the control group (1.01±0.05) ( P<0.01). The relative expression level of DC-STAMP in RAW264.7 cells was 1.92±0.12, which was significantly higher than that of the control group (0.98±0.08) ( P<0.01). Conclusions:The double-layer bone-on-a-chip containing bone matrix is compact in structure, can be cultured in vitro for a long time, has good biocompatibility and can be used for inducing osteogenic and osteoclast differentiation. Therefore, it is expected to provide a new research platform for exploring the mechanism of osteoporosis and medication screening.
ABSTRACT
In recent years, advancements in microfabrication technology and tissue engineering have propelled the development of a novel platform known as organoid-on-a-chip for drug screening and disease modeling. This platform integrates organoids and organ-on-a-chip technologies, emerging as a promising approach for in vitro modeling of human organs. Organ-on-a-chip leverages microfluidic device to simulate the physiological environment of specific organs, offering a more dynamic and flexible setting that can mimic a more comprehensive human biological context. However, the lack of functional vasculature has remained a major challenge in this technology. Vascularization is crucial for the long-term cultivation and in vitro modeling of organoids, which is of great significance in drug development and personalized medical approaches. The authors reviewed the research progress in the construction of vascularized organoid-on-a-chip including the methods for constructing in vitro vascularized models, vascularization of organoids, etc, which may serve as a reference for the construction of fully functional vascularized organoid-on-a-chip.
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.