A high-throughput biomimetic bone-on-a-chip platform with artificial intelligence-assisted image analysis for osteoporosis drug testing.

Although numerous organ-on-a-chips have been developed, bone-on-a-chip platforms have rarely been reported because of the high complexity of the bone microenvironment. With an increase in the elderly population, a high-risk group for bone-related diseases such as osteoporosis, it is essential to develop a precise bone-mimicking model for efficient drug screening and accurate evaluation in preclinical studies. Here, we developed a high-throughput biomimetic bone-on-a-chip platform combined with an artificial intelligence (AI)-based image analysis system. To recapitulate the key aspects of natural bone microenvironment, mouse osteocytes (IDG-SW3) and osteoblasts (MC3T3-E1) were cocultured within the osteoblast-derived decellularized extracellular matrix (OB-dECM) built in a well plate-based three-dimensional gel unit. This platform spatiotemporally and configurationally mimics the characteristics of the structural bone unit, known as the osteon. Combinations of native and bioactive ingredients obtained from the OB-dECM and coculture of two types of bone cells synergistically enhanced osteogenic functions such as osteocyte differentiation and osteoblast maturation. This platform provides a uniform and transparent imaging window that facilitates the observation of cell-cell interactions and features high-throughput bone units in a well plate that is compatible with a high-content screening system, enabling fast and easy drug tests. The drug efficacy of anti-SOST antibody, which is a newly developed osteoporosis drug for bone formation, was tested via ß-catenin translocation analysis, and the performance of the platform was evaluated using AI-based deep learning analysis. This platform could be a cutting-edge translational tool for bone-related diseases and an efficient alternative to bone models for the development of promising drugs.

Enhanced osteogenic differentiation of human mesenchymal stem cells using size-controlled graphene oxide flakes.

Recently, it has been revealed that the physical microenvironment can be translated into cellular mechanosensing to direct human mesenchymal stem cell (hMSC) differentiation. Graphene oxide (GO), a major derivative of graphene, has been regarded as a promising material for stem cell lineage specification due to its biocompatibility and unique physical properties to interact with stem cells. Especially, the lateral size of GO flakes is regarded as the key factor regulating cellular response caused by GO. In this work, GO that had been mechanically created and had an average diameter of 0.9, 1.1, and 1.7 m was produced using a ball-mill process. When size-controlled GO flakes were applied to hMSCs, osteogenic differentiation was enhanced by GO with a specific average diameter of 1.7 µm. It was confirmed that osteogenic differentiation was increased due to the enhanced expression of focal adhesion and the development of focal adhesion subordinate signals via extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MEK) pathway. These results suggest that size-controlled GO flakes could be efficient materials for promoting osteogenesis of hMSCs. Results of this study could also improve our understanding of the correlation between hMSCs and cellular responses to GO.

Enhancement of anti-tumor activity in melanoma using arginine deiminase fused with 30Kc19α protein.

Arginine deiminase (ADI) is a microbial-derived enzyme which catalyzes the conversion of L-arginine into L-citrulline. ADI originating from Mycoplasma has been reported to present anti-tumor activity against arginine-auxotrophic tumors, including melanoma. Melanoma cells are sensitive to arginine depletion due to reduced expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis. However, clinical applications of recombinant ADI for melanoma treatment present some limitations. Since recombinant ADI is not human-derived, it shows instability, proteolytic degradation, and antigenicity in human serum. In addition, there is a problem of drug resistance issue due to the intracellular expression of once-silenced ASS1. Moreover, recombinant ADI proteins are mainly expressed as inclusion body forms in Escherichia coli and require a time-consuming refolding process to turn them back into active form. Herein, we propose fusion of recombinant ADI from Mycoplasma hominis and 30Kc19α, a cell-penetrating protein which also increases stability and soluble expression of cargo proteins, to overcome these problems. We inserted matrix metalloproteinase-2 cleavable linker between ADI and 30Kc19α to increase enzyme activity in melanoma cells. Compared to ADI, ADI-LK-30Kc19α showed enhanced solubility, stability, and cell penetration. The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment. KEY POINTS: • Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli • 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active • Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine.

Prevention of collagen hydrogel contraction using polydopamine-coating and alginate outer shell increases cell contractile force.

Collagen is the most abundant protein in the extracellular matrix of mammals and has a great effect on various cell behaviors including adhesion, differentiation, and migration. However, it is difficult to utilize collagen gel as a physical scaffold in vitro because of its severe contraction. Decrease in the overall hydrogel volume induces changes in cell distribution, and mass transfer within the gel. Uncontrolled mechanical and physiological factors in the fibrous matrix result in uncontrolled cell behaviors in the surrounding cells. In this study, two strategies were used to minimize the contraction of collagen gel. A disk-shaped frame made of polydopamine-coated polydimethylsiloxane (PDMS) prevented horizontal contraction at the edge of the hydrogel. The sequentially cross-linked collagen gel with alginate outer shell (CA-shell) structure inhibited the vertical gel contraction. The combined method synergistically prevented the hydrogel from shrinkage in long-term 3D cell culture. We observed the shift in balance of differentiation from adipogenesis to osteogenesis in mesenchymal stem cells under the environment where gel contraction was prevented, and confirmed that this phenomenon is closely associated with the mechanotransduction based on Yes-associated protein (YAP) localization. Development of this contraction inhibition platform made it possible to investigate the influence of regulation of cellular microenvironments. The physical properties of the hydrogel fabricated in this study were similar to that of pure collagen gel but completely changed the cell behavior within the gel by inhibition of gel contraction. The platform can be used to broaden our understanding of the fundamental mechanism underlying cell-matrix interactions and reproduce extracellular matrix in vivo.

Partially Digested Osteoblast Cell Line-Derived Extracellular Matrix Induces Rapid Mineralization and Osteogenesis.

An extracellular matrix (ECM) utilized as a biomaterial can be obtained from organs of living organisms. Therefore, it has some limitations in its supply because of insufficient organs. Furthermore, therapeutic efficacy of ECMs varies depending on factors such as donor's health condition and age. For this reason, ECMs obtained from a cell line could be a good alternative because they can be produced under a controlled environment with uniform quality. Thus, the purpose of this study was to investigate the potential of the MC3T3-E1 cell line-derived ECM as bone graft. The optimized decellularization process was developed to separate the ECM from MC3T3-E1, osteoblast cell line, using Trypsin-EDTA and Triton X-100. The decellularized ECM was partially digested using pepsin. Also, human bone marrow-derived mesenchymal stem cells induced faster osteogenesis on the ECM-coated surface than on the collagen-coated surface. Partially digested ECM fragments were embedded on the polyethylene glycol scaffold without additional chemical modification or crosslinking. Micro-computed tomography and histological analysis results showed that the ECM in the scaffold promoted actual bone regeneration after in vivo implantation to a mouse calvarial defect model. This study suggests that the bone-specific ECM derived from the cell line can replace the ECM from organs for application in tissue engineering and regenerative medicine.