Frontiers in Bioengineering and Biotechnology最新文献

Post-traumatic osteoarthritis (PTOA), a subtype of osteoarthritis initiated by joint trauma, is driven by unresolved early inflammation that ultimately leads to cartilage degeneration. Although animal models have advanced our understanding of disease progression, they offer limited resolution of the early molecular events following trauma. In this study, we developed a transwell-based in vitro triculture model mimicking the early joint environment post-injury, incorporating macrophages, fibroblast-like synoviocytes (FLSs), and human articular chondrocytes (HACs). In lieu of the commonly used macrophage activator, lipopolysaccharide (LPS), this study utilizes fibronectin fragments (Fnfs), which belong to the damage-associated molecules released upon trauma to cartilage, to activate macrophages and simulate post-traumatic inflammation. The triculture was maintained for 12 days while promoting paracrine-only communication between the cell types. The activation of macrophages by Fnfs led to a sustained expression of pNFκB in both HACs and FLSs, as shown by immunofluorescence, alongside increased gene expression of inflammatory mediators MMP3, MMP13, and TNF-α. Fnfs triggered catabolic signaling across all joint-resident cell types used in this model. To support the translational relevance of the in vitro findings, a complementary in vivo experiment in which Fnfs were injected intra-articularly showed increased MMP activity gene expression and reduced COL2A1 gene expression in joint cartilage. The cytokine and gene expression profiles observed in the triculture model closely mirrored those observed in early-stage in vivo PTOA models and in the patient-derived synovial fluid obtained in the early traumatic phase, underscoring the model's physiological relevance. This triculture platform captures the key aspects of early PTOA processes driven by macrophage activation and offers a biologically relevant tool for mechanistic studies and therapeutic screening.

[This corrects the article DOI: 10.3389/fbioe.2025.1430222.].

Fluorescent proteins are important reporter tools to investigate biological processes in the cellular environment at the molecular level. The spectrum of available fluorescent proteins has been greatly expanded by red-emitting fluorescent variants such as the commonly used mCherry. However, the presence of alternative translation initiation sites (aTIS) in mCherry allows for production of shorter protein isoforms with different properties that can bias the results of studies in which mCherry intensities are evaluated. In the present study, we used a novel approach of spectroscopic techniques, including Förster resonance energy transfer (FRET) to investigate the impact of aTIS on the photophysical properties and the functionality of mCherry in both, prokaryotic and eukaryotic expression systems. To this aim, FRET tandem constructs with different translation initiation sites, comprising mNeonGreen as donor fluorophore and mCherry as acceptor, were designed and systematically analyzed using steady-state spectroscopy, time- and spectrally-resolved fluorescence measurements, and fluorescence lifetime imaging (FLIM) based FRET measurements. The long isoforms exhibited similar photophysical properties like the full-length mCherry protein. They were also suitable FRET acceptors when coupled to mNeonGreen. The choice of translation initiation site markedly affected donor fluorescence lifetime, fluorescence intensity, and efficiency of energy transfer of the FRET constructs. Longer mCherry isoforms retained FRET acceptor functionality whereas shorter translational isoforms were non-functional, i.e., were non-fluorescent and had no effect on donor fluorescence lifetime. Our results provide insight into the implications of aTIS when using mCherry as fluorescent reporter. Overall, our study highlights the importance of considering translation initiation mechanisms in both pro- and eukaryotic systems, as they can substantially impact protein functionality and the interpretation of biological measurements.

Transient gene expression (TGE) is commonly used for the rapid production of protein-based therapeutics, including antibodies that require post-translational modifications. We previously published a protocol for efficient and cost-effective TGE of multispecific and multivalent antibodies. Here, we describe an optimized version of this protocol with key improvements in cost, workflow speed, and production capacity. First, the expensive Expi293 expression medium was replaced with BalanCD HEK293 medium, resulting in a substantial decrease in medium-related costs by approximately 90%. The addition of Pluronic F-68 was omitted, as the new medium already contains a similar surfactant. To minimize plasmid DNA usage, salmon sperm DNA was included as filler DNA during transfection, enabling a significant reduction in plasmid DNA input without compromising antibody yield. Second, the harvesting procedure was shortened from 2.5 h to just 15 min by adding the mineral compound diatomaceous earth (Celpure®) to the culture supernatant. This effectively absorbs and sequesters cells and debris, allowing rapid filtration without filter clogging or the previously required 1-h centrifugation step. Finally, we recommend high-flow rate HiScreen Fibro PrismA columns to further accelerate downstream antibody purification. Together, these improvements streamline the TGE workflow in Expi293F cells, enhance scalability, and increase throughput while maintaining efficiency in antibody production.

Background: The temporomandibular joint disc plays a vital role in daily activities, and when it is compromised, it significantly impairs oral health and quality of life. The use of animal tissues for decellularized tissue engineering applications has been gaining interest, and an appropriate method for storing these tissues before processing has yet to be explored.

Methods: This study characterizes the native temporomandibular ovine disc and compares storage protocols aimed at maintaining its morphology, biochemical content, and mechanical and thermal properties. Three storage protocols were tested: (i) freezing at -20 °C in phosphate-buffered saline (PBS) and thawing at 4 °C (PBS + 4 °C); (ii) freezing at -20 °C in PBS and thawing at room temperature (RT) (PBS + RT); and (iii) wrapping the discs in PBS-embedded gauze, freezing at -20 °C, and thawing at RT (Gauze + RT). Protocols were evaluated for short-term storage at 1, 7, and 14 days, and compared with a native and a collagenase-treated disc.

Results: All conservation protocols induced changes, though less pronounced than the enzymatic degradation. The PBS + 4 °C and PBS + RT highlighted contrasting biochemical and mechanical outcomes, and thermal analysis revealed alterations to collagen structure. The Gauze + RT protocol preserved the biochemical content over time but exhibited a higher compression modulus on day 14.

Conclusion: These results highlight how crucial it is to select adequate conservation techniques when preparing the TMJ disc for future studies.

Introduction: Electrospun bioactive polymer biomaterials have gained increasing interest as platforms for cartilage tissue engineering due to their ability to mimic the extracellular matrix (ECM) and provide structural and biochemical support for mesenchymal stem cell (MSC) differentiation. The present study aimed to assess the influence of these bioactive compounds on the chondrogenic differentiation of MSCs.

Methods: Poly(L-lactic acid) (PLA) fibrous mats were fabricated using electrospinning techniques, including standard and coaxial electrospinning, to incorporate bioactive components, namely collagen I and platelet-rich plasma (PRP). The wettability, the fibers diameter, degradation of the mats and PRP release profile were assessed. MSC differentiation culture was performed to determine the effect of the mats on the chondrogenic lineage.

Results: The fabricated fibrous mats exhibited distinct morphological and physicochemical characteristics, with core-shell (CS) fibers demonstrating reduced diameters compared to pure PLA and PLA-collagen (Col) fibers. Wettability studies revealed that PRP encapsulation within the PLA shell did not alter the hydrophobic nature of the material, while the presence of collagen significantly enhanced its hydrophilicity. The PRP release profile from CS fibers exhibited a controlled release within the initial 3 days, followed by stabilization. Furthermore, MSC differentiation studies confirmed that both PRP and collagen-enriched fibrous mats supported chondrogenic differentiation over 14-day period, with Col mats demonstrating the highest glycosaminoglycan (GAG) production. The presence of aggrecan, a key chondrogenic marker, was most pronounced on collagen mats and comparable or lower on PRP (CS) compared with PLA, particularly at 14 days.

Discussion: Furthermore, the observations revealed the presence of two critical markers of cartilage differentiation: namely, actin cytoskeletal reorganization and depolymerization. The presented findings highlight the potential of bioactive PLA fibrous mats enriched with PRP and collagen I as promising platforms for cartilage tissue regeneration. The combination of electrospinning techniques enables tailored fiber structures that support chondrogenesis, offering a potential alternative for tissue engineering applications.

Background: This study aims to investigate the application of visual information processing mechanisms in the segmentation of stem cell (SC) images. The cognitive principles underlying visual information processing were analyzed, and the limitations of conventional segmentation methods were evaluated using phase-contrast microscopy images of stem cells.

Methods: An optimized segmentation method incorporating halo correction was developed to address the limitations of traditional approaches. The performance of the proposed method was experimentally validated and compared with existing techniques.

Results: The proposed method achieved segmentation accuracy, recall, precision, and F1-score values of 96.5%, 94.9%, 91.4%, and 93.9%, respectively, outperforming existing approaches. Additionally, the confluency error on the Human Mesenchymal Stem Cells dataset and the C2C12 dataset was 0.07 and 0.05, respectively, indicating superior performance compared to equivalent methods.

Conclusion: The findings demonstrate that the proposed method offers enhanced efficacy for stem cell image segmentation tasks.

Introduction: Globally, more than 17% of human infections are caused by vector-borne viruses, which result in more than 700,000 deaths annually as per the World Health Organization. Mosquitoes and ticks are the primary arthropod vectors, along with sandflies and midges. More than 500 arthropod-borne viruses (arboviruses) have been described, with more than 150 causing human disease. It is important to understand the public health risk associated with arboviruses.

Methods: We used multi-criteria decision analysis (MCDA) techniques and a Decision Support Framework (DSF) employing a logic tree format to identify high-risk arboviruses, applying these approaches to only those arboviruses transmitted by flying insects (i.e., mosquitos, sandflies, and midges) due to their potential for efficient transmission and habitat expansion.

Results: A literature review of 54 arboviruses against 13 criteria was conducted for assessing risk and documenting the findings that support this assessment. The most prominent data gaps found were those for the annual global incidence, the severity of disease, and long-term impact. Technical review of published data and associated scoring recommendations by subject matter experts (SMEs) were found to be critical, particularly for pathogens with very few known cases. The MCDA analysis supported the intuitive sense that agents with high mortality and morbidity rates should rank higher on the relative risk scale when considering disease persistence and severity. However, comparing scores to suggest thresholds for designating high risk versus (vs) moderate risk vs low risk, was challenging and will require additional real time data during an outbreak. The DSF utilized a logic tree approach to identify arboviruses that were of sufficiently low enough concern that they could be ruled out from further consideration. In contrast to the MCDA approach, the DSF ruled out an arbovirus if it failed to meet even one criteria threshold.

Conclusion: The MCDA and DSF approaches arrived at similar conclusions, suggesting that using these analytical approaches for an arbovirus risk assessment added robustness for decision making.

Background: Foot and ankle diseases significantly impact quality of life, with regenerative medicine emerging as a promising approach. A comprehensive evaluation of both efficacy and safety is paramount for its clinical translation.

Methods: A comprehensive literature search was conducted in PubMed using keywords "regenerative medicine" and "foot and ankle" (as of 31 December 2024). Studies were categorized by technology and disease.

Results: PRP and HA showed short-term efficacy in talar cartilage repair; stem cells enhanced functional recovery in ankle osteoarthritis. 3D printing enabled personalized implants. Exosomes and AI were identified as future directions. However, the reporting of safety data was often sporadic and non-standardized, highlighting the need for more systematic monitoring in future studies.

Conclusion: Regenerative therapies demonstrate potential but require further validation through robust trials that prioritize standardized safety reporting alongside efficacy outcomes. Gaps in exosome isolation, long-term safety, and clinical translation need addressing.