Construction of double-layer bone-on-a-chip containing bone matrix / 中华创伤杂志

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.

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

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.

Implications on the Development of Animal Disease Models from FDA Modernization Act 2.0 / 实验动物与比较医学

Laboratory animals are the foundational conditions and indispensable technical support in life science research and biomedical industry development. The scientific development of animal models of diseases is of great significance to biomedical research and industrial development. In light of the booming development of multiple emerging in vitro modelling technologies over the past decade, in 2022, the U.S. Senate unanimously passed the bill FDA Modernization Act 2.0. This bill rescinded the requirement for animal testing in investigating the safety and effectiveness of a drug—a federal mandate since 1938, and highlighted the potential of various in vitro disease modeling approaches in future biomedical fields. This paper provides a comprehensive review of the latest advances and applications of in vitro disease modeling approaches in academia and industry followed by an interpretation of the FDA bill, namely cell culture, organoid, organ-on-a-chip, 3D bio-printing model and computer-based model. The paper next introduces the crossed applications of various disease models and discusses the advantages and disadvantages of each system, thereby providing insights into future trends in the use of animal disease models in China.

Application of vascularized organ-on-a-chip in simulating physiological and pathological processes / 中国药科大学学报

@#With the development of biomimetic technology, more and more in vitro models are used to simulate human physiological and pathological processes.These in vitro models can solve some scientific problems, such as studying drug effects in real-timely and visually.As an in vitro model, organ-on-a-chip provides novel means and methods for basic and applied science.The vascularized organ-on-a-chip, as a special kind of organ-on-a-chip, can better simulate the structure and function of human blood vessels.In this review, we summarized the structure and function of different vascularized organ-on-a-chip, analyzed the application of vascularized organ-on-a-chip in simulating physiological and pathological processes, and discussed the advantages and problems to be solved of vascularized organ-on-a-chip as a new in vitro model.Finally, the application of vascularized organ-on-a-chip is proposed.

Frontier hotspots and research progress on 3D bioprinting in organ reconstruction / 器官移植

3D bioprinting is an advanced manufacturing technology that utilizes biomaterials and bioactive components to manufacture artificial tissues and organs. It has been widely applied in multiple medical fields and possesses outstanding advantages in organ reconstruction. In recent years, 3D bioprinted organs have made an array of groundbreaking achievements. Nevertheless, it is still in the exploratory stage of research and development and still has bottleneck problems, which can not be applied in organ transplantation in vivo. In this article, the application of 3D printing technology in medicine, characteristics of 3D bioprinting technology, research hotspots and difficulties in bionic structure, functional reconstruction and immune response of 3D bioprinted organs, and the latest research progress on 3D bioprinting technology were illustrated, and the application prospect of 3D bioprinting technology in the field of organ reconstruction was elucidated, aiming to provide novel ideas for the research and clinical application of organ reconstruction and artificial organ reconstruction, and promote the development of organ transplantation and individualized medicine.

Microchip based on 3D printing technology for simulating intestinal villi and tumor surface topography / 上海交通大学学报（医学版）

Objective: To propose a method for making organ-on-a-chip based on 3D printing, and study the relationship between cell growth on the chips and various factors. Methods: Through 3D printing technology and surface microstructure transfer method, ulcer-like and ridge-like mi-crostructures of the tumor surface and the intestinal villi were fabricated on a polydimethylsiloxane (PDMS) chip. Combined with fluorescence imaging, the effects of surface modification, shapes and heights of microstructures, and culture time on the surface coverage and density of Caco-2 cells on the chip were measured. Results:The PDMS chip was more likely to induce cell adhesion and growth rather than the 3D printing resin chip. On the surface of three-dimensional structure, cell surface coverage and cell density increased after the surface was treated with rat tail collagen (P0.05). Conclusion: The intestinal villi and tumor topological organ chips can be fabricated by 3D printing technology and surface microstructure transfer method. The surface modification and microstructure height affect the cell growth on the surface.

Organ-on-a-chip and the kidney

Traditional approaches to pathophysiology are advancing but still have many limitations that arise from real biologic systems and their associated physiological phenomena being too complicated. Microfluidics is a novel technology in the field of engineering, which provides new options that may overcome these hurdles. Microfluidics handles small volumes of fluids and may apply to various applications such as DNA analysis chips, other lab-on-a-chip analyses, micropropulsion, and microthermal technologies. Among them, organ-on-a-chip applications allow the fabrication of minimal functional units of a single organ or multiple organs. Relevant to the field of nephrology, renal tubular cells have been integrated with microfluidic devices for making kidneys-on-a-chip. Although still early in development, kidneys-on-a-chip are showing potential to provide a better understanding of the kidney to replace some traditional animal and human studies, particularly as more cell types are incorporated toward the development of a complete glomerulion-a-chip.