Journal of Biological Engineering最新文献

Bio-hybrid micromotors, active structures composed of both biological and synthetic components, are promising for use in several biomedical applications including targeted drug delivery, tissue engineering, and biosensing. Among biological candidates, erythrocytes are well suited for use as the biological component of bio-hybrid micromotors due to their biocompatibility, mechanical deformability, and long circulation time. However, their symmetric shape and small size make controlled actuation of these devices particularly challenging. Here, we present a novel strategy to overcome these limitations by fabricating achiral erythrocyte micromotors with enhanced propulsion efficiency. Inspired by recent work on synthetic achiral microswimmers, we report two and three-cell micromotors fabricated through biotin-streptavidin binding. These self-assembled red blood cell (RBC) structures are then interfaced with magnetic beads enabling them to swim and roll under the propulsion of a single homogenous rotating magnetic field at a much greater velocity compared to single cell micromotors in both Newtonian and viscoelastic fluids. Further, to demonstrate biomedical application of these self-assembled micromotors, the chemotherapeutic agent doxorubicin is loaded into RBC achiral micromotors, which are magnetically directed to cancer cells within a microfluidic chamber, successfully delivering their anticancer payload. The fabrication and propulsion method reported here will aid in the development of future erythrocyte-based micromotors for drug delivery and cancer therapy.

Polycystic ovary syndrome (PCOS), the most common endocrine disease in women of reproductive age, severely impacts female fertility due to chronic anovulation and currently lacks effective clinical treatment strategies. The extracellular matrix (ECM) is a three-dimensional, non-cellular framework comprising molecules such as collagens, elastin, and laminin, which support the ovarian structure and provide extracellular signals to cells. Changes in ECM localization and composition can disturb local biochemical pathways, impair folliculogenesis, and reduce the fertility of women. This paper explores innovative therapeutic approaches for PCOS by investigating the mechanisms underlying PCOS pathogenesis due to ECM dysregulation. This includes ECM deposition-induced inflammation and fibrosis, impaired ECM degradation, altered mechanical forces in ECM remodeling, and disrupted interactions between granulosa cells and the ECM. In the second part, we present therapeutic strategies informed by these pathogenic mechanisms, integrating insights from basic and clinical research. More importantly, this paper introduces innovative therapies for POCS that regulate ECM. These therapeutic strategies represent future development directions. In the final section, we summarize the advantages, potential challenges, and prospects of ECM-based treatments for improving fertility in PCOS. Overall, this review underscores the emerging significance of ECM-targeted interventions in unraveling PCOS pathophysiology and paves the way for the development of more precise and effective fertility-preserving therapies.

Background: Nanomaterials offer increasing applications across diverse sectors, including food science, medicine, and electronics. Environmental risk assessment is crucial for ensuring the safety and sustainability of nanomaterials. However, high-throughput screening (HTS) of their potential toxicity remains challenging owing to their unique physicochemical properties.

Results: This study introduces a novel pulmonary three-dimensional (3D) floating extracellular matrix (ECM) model utilizing a 384-pillar/well platform for HTS of nanotoxicity. Compared with conventional HTS models based on two-dimensional (2D) cells, the 3D model developed in this study successfully addressed the issues related to the aggregation and sedimentation of nanoparticles and their possible optical interference with the toxicity assays. Using 20 nm silica nanoparticles (SiNPs), we assessed cell viability and nanoparticle uptake in both serum-containing and serum-free culture media. While the 2D model showed high SiNPs toxicity regardless of the media composition, the pulmonary 3D floating ECM model demonstrated variable toxicities that depended on the SiNPs behaviors under different conditions.

Conclusions: By reducing the uncertainties associated with the sedimentation and optical interference of nanomaterials, our 3D model provided a more precise analysis of cytotoxicity. This study highlights the potential of using new approach methodologies and improved HTS approaches to enhance the efficiency and accuracy of risk assessment protocols for emerging nanomaterials.

Background: Halogenation plays a crucial role in enhancing the properties of small molecules, particularly by making them more effective for applications in agrochemicals and pharmaceuticals. Notably, approximately a quarter of current pharmaceuticals are halogenated. While chemical halogenation remains the most widely employed method for producing halogenated molecules, it has significant drawbacks, including extreme reaction conditions, heavy pollution, and the use of toxic reagents. In contrast, bio-halogenation offers a "greener" approach to generating halogenated compounds. However, its industrial application is limited due to the low activity and stability of naturally occurring halogenase enzymes.

Results: In this study, we identified a novel tryptophan halogenase, SsDiHal, from Saccharothrix sp. NRRL B-16348 through genome mining. We found that SsDiHal catalyzes a two-step chlorination of tryptophan to sequentially yield 7-chlorotryptophan and 6,7-dichlorotryptophan, making SsDiHal the first naturally occurring tryptophan dihalogenase to be identified. Using a strcutral model of SsDiHal to guide mutagensis, several SsDiHal mutants were generated and tested for improved catalytic efficiency and altered regioselectivity. Compared to the halogenation activity of the wild type SsDiHal, the V53I, V53I/I83V and N470S mutants demonstrated significantly enhanced catalytic efficiency, with 7.7-, 4.16-, and 7.4-fold increases respectively, for the L-tryptophan substrate. While no change in regioselectivity was observed for the V53I, I83V, F112Y, and V53I/I83V mutants, a notable regioselectivity shift was found in the N470S mutant. Specifically, this mutant synthesized 6-chlorotryptophan as the first product, rather than the canonical 7-chlorotryptophan that is synthesized by wild type SsDiHal with no effect in its dihlogenation function.

Conclusion: Overall, this work not only adds a novel dihalogenase to the growing field of halogenating enzymes but also demonstrates that leveraging a structrual model to guide engineering of halogenases can both enhance the catalytic efficiency and modify regioselectivity of the wild type enzyme. This work holds significant potential for green applications in the agrochemical and pharmaceutical industries.

Bone defects resulting from trauma, infection, or surgical resection require biomaterials that support osteogenesis and vascularization for effective regeneration. In this study, we developed a 3D-printed magnesium- and strontium-co-doped calcium silicate (MSCS) scaffold using direct ink writing to optimize its bioactivity and structural integrity. X-ray diffraction confirmed the successful incorporation of Sr and Mg, leading to phase modifications that influenced ion release and degradation. Wettability and mechanical testing showed that Sr improved the stability, while Mg accelerated degradation, with M5S5 co-doping exhibiting a balanced degradation profile. In vitro, Wharton's jelly mesenchymal stromal cells cultured on M5S5 scaffolds displayed enhanced proliferation, cytoskeletal organization, and osteogenic differentiation, as evidenced by increased alkaline phosphatase activity and bone matrix protein expression. Angiogenesis assays using human umbilical vein endothelial cells revealed that Sr and Mg co-doping synergistically enhanced vascular endothelial growth factor and angiopoietin-1 secretion, thereby promoting endothelial tube formation. In vivo micro-computed tomography and histological analysis of a rabbit femoral defect model confirmed that M5S5 facilitated extensive new bone formation, exhibiting superior trabecular architecture and mineralization. These findings highlight MSCS scaffolds as promising biomaterials for bone tissue engineering applications.

This article presents perspectives on the need to transition from the current unsustainable consumptive fossil-based linear (take-make-use-dispose) systems that produces huge quantities of wastes, pollutes land, water and air, and contributes to climate change to sustainable bio-based circular (take-make-use-decay-reuse) systems. In the article, the word 'fossil' refers to all forms of mined carbon and minerals from the Earth, including water from aquafers, which cannot be replenished at the rate that will maintain their capacity to provide for the future. The natural world through its many circular systems uses energy and renewable resources to perform functions that produce zero waste. One organism's waste becomes another organism's food, material, and energy, forming a circular loop (take-make-use-decay-reuse). Over the past 4 years, deliberate engagements with leaders of multiple disciplines and stakeholders resulted in conclusions that the problems of the complex biologically active systems (biosystems) that are intertwined with natural systems and socio-economic systems can only be addressed by having a robust culture of convergent science and engineering and systems-thinking for transitioning from linear fossil-based to circular bioeconomy systems. We present the need and propose forming a multidisciplinary professional society alliance to promote and support networks of multidisciplinary teams to address problems of complex, intertwined bio-natural-socio-economic systems of systems. This article proposes that the Institute of Biological Engineering (IBE), a society whose primary objective is to "to apply biology-inspired engineering principles to design systems to improve the quality of the human condition", and inculcates a culture of convergent science and engineering that has members representing expertise of multiple science and engineering discipline, is potentially an excellent candidate to play a pivotal role in designing innovative solutions for advancing sustainable circular bioeconomy systems.

Background: Precise and dynamic transcriptional regulation is a cornerstone of synthetic biology, enabling the construction of robust genetic circuits and programmable cellular systems. However, existing regulatory tools are often limited by issues such as leaky transcription and insufficient tunability, particularly in high-expression or complex genetic contexts. This study aimed to develop a CRISPRi-aided genetic switch platform that overcomes these limitations and expands the functionality of transcriptional regulation tools in synthetic biology.

Results: We established a versatile CRISPRi-aided genetic switch platform by integrating transcription factor-based biosensors with the Type V-A FnCas12a CRISPR system. Exploiting the RNase activity of FndCas12a, this system processes CRISPR RNAs (crRNAs) directly from biosensor-responsive mRNA transcripts, enabling precise, signal-dependent transcriptional regulation. To mitigate basal transcription and enhance regulatory precision, transcriptional terminator filters were incorporated, reducing leaky expression and increasing the dynamic range of target gene regulation. The platform demonstrated exceptional adaptability across diverse applications, including ligand-inducible genetic switches for transcriptional control, signal amplification circuits for enhanced output, and metabolic genetic switches for pathway reprogramming. Notably, the metabolic genetic switch dynamically repressed the endogenous gapA gene while compensating with orthologous gapC expression, effectively redirecting metabolic flux to balance cell growth.

Conclusions: The CRISPRi-aided genetic switch provides a powerful and flexible toolkit for synthetic biology, addressing the limitations of existing systems. By enabling precise and tunable transcriptional regulation, it offers robust solutions for a wide array of biotechnological applications, including pathway engineering and synthetic gene networks.

The convergence of cell-free protein synthesis (CFPS) and vesicle-based delivery platforms presents a promising avenue for therapeutic development. The open environment of CFPS offers precise control over protein synthesis by enabling the modulation of synthetic conditions. Additionally, vesicle-based platforms provide enhanced stability, bioavailability, and targeted delivery. This synergy facilitates the efficient production of complex proteins-including membrane proteins, antibody fragments, and proteins requiring post-translational modifications (PTMs)-and supports novel drug delivery strategies. While existing reviews have covered synthetic cells and biomanufacturing broadly, a dedicated analysis of CFPS system-containing vesicles (CFVs) for therapeutic applications remains absent from the literature. This review addresses this knowledge gap by providing a comprehensive examination of CFVs, highlighting their potential as programmable drug delivery platforms through the integration of genetic circuits. It emphasizes the advantages of CFPS over traditional cell-based approaches and explores the synergistic benefits of combining CFPS with various vesicle systems. These systems offer dynamic control over therapeutic protein production and targeted delivery, enabling precise responses to specific signals in complex environments. Although challenges such as low protein yield and imperfect targeting remain, potential optimization strategies are discussed. This analysis highlights the significant potential of integrating CFPS and vesicle-based delivery to advance biomanufacturing, therapeutic development, and synthetic cell systems, thereby opening new avenues in medicine and healthcare.

Background: Soft-tissue reconstruction is crucial in fields such as plastic surgery and oncology to address the repair of damaged tissues. Knitted scaffolds from bioresorbable copolymers, specifically poly(D,L-lactide) (PLA) and polycaprolactone (PCL), offer mechanical and biological properties that are essential for tissue engineering. This study assessed three-dimensional knitted scaffolds fabricated from melt-spun PLA and PCL multifilaments for soft tissue engineering applications. It examined the impact of the PLA/PCL ratio on the knitted scaffold structure, mechanical properties, and biological responses to determine the optimal composition for adipose tissue reconstruction.

Results: Knitted scaffolds fabricated with the PLA/PCL blends (PLA₇₀/PCL₃₀ and PLA₉₀/PCL₁₀) exhibited distinct mechanical and biological profiles. PLA₇₀/PCL₃₀ scaffolds with a higher PCL content showed enhanced elasticity and porosity, whereas PLA₉₀/PCL₁₀ scaffolds maintained better structural integrity and stiffness. Biological assays confirmed the biocompatibility of all scaffolds in vitro, with no cytotoxic effects. The scaffolds supported adipogenic differentiation in vitro, although PLA₇₀/PCL₃₀ exhibited slightly reduced efficacy. Vascularization was evident using chorioallantoic membrane assays, in which blood vessel formation and penetration were observed, regardless of the scaffold composition. In vivo implantation in rat models revealed effective adipocyte integration, structural stability, and minimal inflammatory response, with PLA₉₀/PCL₁₀ scaffolds outperforming PLA₇₀/PCL₃₀ in terms of vascularization and less macrophage infiltration of connective tissue.

Conclusion: PLA/PCL knitted scaffolds offer a promising solution for enhancing graft volume maintenance and improving long-term outcomes, with tunable mechanical properties and biodegradability. The PLA₉₀/PCL₁₀ scaffold is a superior candidate for adipose tissue reconstruction, balancing the structural stability with biological compatibility. These findings underscore the potential of PLA/PCL scaffolds for reconstructive surgery. Future studies should focus on scalability and long-term biocompatibility to facilitate clinical translation.