Frontiers in Bioengineering and Biotechnology最新文献

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system has significant potential in biological diagnostics because of its precise nucleic acid identification abilities. Traditional CRISPR diagnostics, however, have limitations such as insufficient signal output, dependence on exogenous enzymes, and high equipment demands. Nanozymes, as nanomaterials with enzyme-mimetic catalytic activity, integrate the catalytic efficiency of natural enzymes with the stability and modifiability of nanomaterials, providing a viable resolution to the limitations in CRISPR diagnostics. This article comprehensively evaluates the advancements in nanozyme-enhanced CRISPR diagnostic technologies. Furthermore, it delineates the fundamental attributes of the CRISPR diagnostic system and nanozymes, as well as the necessity of their integration. Moreover, the coupling mechanisms between the CRISPR/Cas system and nanozymes, including the regulation of nanozyme catalytic activity by Cas protein function and CRISPR signal amplification facilitated by nanozymes, were also comprehensively evaluated. The application of this technique in detecting nucleic acid and non-nucleic acid targets was assessed. Further, this study discusses the current limitations of this technology, such as complex separation of heterogeneous systems, laborious reaction protocols, and slow detection rates. The future advancements, such as the establishment of homogenous systems, the creation of integrated devices, and the utilization of single-atom nanozymes, have also been discussed in this review. The results of this study will provide references for the comprehensive integration of nanozymes and CRISPR technology, together with their diagnostic applications.

Introduction: Codon optimization is utilized in biologics design to maximize protein expression. Selecting the host organism's most frequently used codons for each amino acid can significantly enhance recombinant protein expression yields. However, non-optimal codons in mRNA can be critical for functional protein production through inducing pauses in or attenuating protein translation.

Methods: In our study, we have investigated the effect of deoptimizing serine codons in biologics by shifting them from the five most frequently used codons to the least (TCG). Rare serine codons were strategically inserted into the coding sequences of the constant regions in a trispecific antibody (Protein 1), a bispecific antibody (Protein 2), and multiple non-proprietary bispecific antibodies.

Results: We observed that inserting 1-2 rare serine codons within an open reading frame led to expression changes that reduced the formation of mispaired 2x light chain and half-molecule species. Protein purity was drastically increased by incorporating two deoptimized serine codons into a single chain. Notably, we observed a negative correlation between total protein expression yield and final product purity.

Discussion: Taken together, our work demonstrates that incorporation of deoptimized serine codons into a single chain can significantly influence multispecific biologic pairing and enhance final product purity. Our findings align with existing literature showing that rare codon usage modulates translation kinetics and protein folding. Future investigation is warranted to enable a priori identification of the rate-limiting chain in multispecific biologics, thereby guiding strategic codon deoptimization prior to expression.

Introduction: Cardiovascular diseases are a leading cause of mortality, and artificial blood vessels as an alternative strategy are extensively used in clinical settings. Due to the underlying potential for thrombus formation and intimal hyperplasia, the clinical applications of small-caliber (<6 mm) artificial vessels are limited. Promoting rapid endothelialization and enhancing anticoagulant ability are pivotal approaches to achieve long-term patency of small-caliber artificial vessels.

Methods: Biocompatible PCL-ECd nanofibers with a core-shell structure were prepared using coaxial electrospinning. PCL served as the shell layer providing mechanical support, while 30% ECd formed the core layer, accelerating endothelialization. Additionally, incorporating 10% heparin into the core layer endows the P-E/H nanofibers with the desired anticoagulant properties. Coaxial-emulsion electrospinning enables sustained release of ECd and heparin from P-E/H. Finally, the in vitro patency of 4 mm diameter P-E/H vascular scaffolds was evaluated using a closed-loop system.

Results: P-E/H nanofibers exhibited enhanced endothelial cell proliferation, superior hemocompatibility, and ideal anticoagulant properties. The in vitro blood flow patency of a 4 mm diameter P-E/H vascular scaffold indicated the absence of any clot or thrombus.

Conclusion: This study proposed a new strategy for developing small-caliber vascular scaffolds with enhanced hemocompatibility and sustained anticoagulant activity.

Currently, adeno-associated virus (AAV) is one of the most reliable carrier for gene delivery in both proliferating and non-proliferating cells. Stable and long-lasting transgene expression has made this viral vector a key platform for the development of advanced therapy. Nevertheless, the widespread clinical use of AAV-based drugs remains limited due to their immunogenicity, low capsid capacity, and restricted tissue tropism. Tissue tropism depends largely on the the transduction efficiency of AAV capsids. In this study, we modified the standard three-plasmid transfection protocol to provide independent expression of VP1 or VP2 proteins from separate plasmids. Adjusting the ratio of these plasmids in the transfection mixture enabled alteration of the stoichiometric composition of the capsids, as SDS-PAGE and mass spectrometry confirmed. Increasing the amount of VP1 or VP2 in the capsid composition enhanced transduction efficiency, as demonstrated in vitro experiments on HEK293 cells. Obtained results contribute to a more comprehensive understanding of the AAV biology and have perspective of application in gene therapy.

Silicone - namely polydimethylsiloxane (PDMS) - is a widely recognized elastomeric biomaterial commonly used in implantable medical devices such as breast implants, cardiac pacemakers and drug delivery devices. Despite its widespread use, PDMS can elicit a strong foreign body response with fibrous encapsulation that leads to discomfort, pain and implantable device failure in approximately 10% of cases. Poly (styrene-block-isobutylene-block-styrene) (SIBS) is a thermoplastic elastomer used clinically as a drug-eluting coating for coronary stents and experimentally in ocular drainage devices. Although SIBS has demonstrated excellent biocompatibility in these applications, the foreign body response it elicits has not yet been extensively studied in more inflammation-prone anatomic sites such as skin rich in macrophages and fibroblasts. Here, we characterize the physicochemical properties of SIBS, examine its effect on macrophage-fibroblast interactions and evaluate its biocompatibility by implanting it subcutaneously in mice to ultimately assess its viability as a potential alternative to PDMS. We establish that both materials have comparable physicochemical properties, demonstrate that fibroblasts adopt a less contractile pro-inflammatory phenotype when exposed to SIBS-macrophage conditioned media and show reduced fibrotic encapsulation around SIBS implants in mice. These results suggest that SIBS could potentially be a favorable biomaterial alternative to silicone in clinical applications.

Background: Sequential distalization of the maxillary dentition is widely used to gain space for crowding relief, molar relationship correction, and facial profile improvement. Using three-dimensional finite element analysis, we evaluated the biomechanical impact of different distal-end clear aligner designs during premolar distalization in maxillary sequential distalization, with the aim of reducing mesial relapse of teeth that had already reached their planned positions and improving overall distalization efficiency.

Material and methods: Two initial models were constructed: In Model A, both maxillary molars were distalized by 2 mm to reach the planned positions. In Model B, both maxillary molars and the maxillary second premolar were distalized by 2 mm to reach the planned positions. Four aligner configurations were defined in Model A: A0, control (conventional design); A1, distal coverage removed at U7; A2, distal coverage removed at U6; and A3, distal coverage removed at U7 and U6. Five aligner configurations were defined in Model B: B0, control (conventional design); B1, distal coverage removed at U7; B2, distal coverage removed at U5 and U6; B3, distal coverage removed only at U5; and B4, distal coverage removed at U7, U6, and U5. We quantified tooth displacement, space-closure component ratios, and periodontal ligament (PDL) equivalent stress under each condition.

Results: For Model A, the contribution of U5 distal displacement to U5-U6 space closure was 68.08%, 66.48%, 70.44%, and 92.01% in A0-A3, respectively, and was highest in A3. In Model B, the contribution of U4 distal displacement to U4-U5 space closure was 69.22%, 68.15%, 70.55%, 70.47%, and 81.19% in B0-B4, respectively, and was highest in B4.

Conclusion: During second premolar distalization in maxillary sequential distalization, removing distal-portion coverage at U7 and U6 effectively reduced mesial relapse of already distalized molars, thereby protecting posterior anchorage. Similarly, during first premolar distalization, to reduce relapse of distalized teeth, distal-portion coverage should be removed at U7, U6, and U5.

Objective: Interstitial fluid flow within the osteonal lacunar-canalicular system (LCS) is crucial for osteocyte mechanotransduction and bone remodeling. This study aims to develop a three-dimensional finite element model of an osteon with gradient-varying boundary conditions to systematically investigate how mechanical loading, outer wall constraints, and pulsatile blood pressure modulate intra-osteonal fluid flow.

Methods: This study constructs a three-dimensional finite element model to systematically analyze the dynamic responses of fluid flow behavior under gradient boundary conditions. Gradient parametric analyses were performed by varying: (1) axial strain amplitudes (250-5000 με) to simulate different activity levels; (2) radial displacement constraints at the outer wall (0- 0.042 μm) to represent confinement by surrounding tissues; and (3) pulsatile blood pressure amplitudes (A = 0-2.5) at the inner wall to mimic physiological to hypertensive conditions. The resulting pore pressure, fluid velocity, and fluid shear stress (FSS) distributions were analyzed.

Results: All parameters exhibited axisymmetric distributions. Peak pore pressure, fluid velocity, and FSS increased nearly linearly with strain magnitude, ranging from 1.7×10⁴ to 1.4×10⁵ Pa, 1.69×10^-8 to 3.50×10^-8 m/s, and 0.34 to 6.5 Pa, respectively. Relaxation of outer wall constraints from fully constrained (0 μm) to fully elastic (0.042 μm) significantly reduced all three parameters. Elevated pulsatile blood pressure markedly increased intra-osteonal pore pressure (from 2.7×10⁴ to 6.5×10⁴ Pa) but had minimal effect on velocity and FSS. A subsequent multiscale validation using an explicit LCS model showed that the macro-scale poroelastic model accurately captures global trends, while local FSS within canaliculi is amplified by a factor of 1.5-2.5.

Conclusion: The gradient boundary condition approach effectively quantifies the differential and synergistic effects of mechanical load, structural constraint, and vascular pressure on the osteonal fluid environment. These findings provide a quantitative framework for understanding mechanotransduction in bone and may inform clinical strategies for managing bone adaptation and disease.

Metabolic bone diseases (MBDs), such as osteoporosis and rickets, present significant clinical challenges due to the chronic nature of treatment and the limitations of conventional systemic therapies. Oral medications often suffer from low bioavailability and gastrointestinal intolerance, while injectable biologics are hampered by poor patient adherence. Microneedle (MN) systems have emerged as a transformative transdermal delivery platform capable of overcoming these barriers. This review provides a comprehensive overview of MN technology, detailing its classification, material properties, and advantages in bypassing the stratum corneum for painless administration. We analyze how MNs have evolved from physical conduits into intelligent therapeutic platforms that integrate bone-targeting ligands, stimuli-responsive release mechanisms, and immunomodulatory functions to precisely regulate the bone microenvironment. Furthermore, we summarize recent preclinical advances in MN applications for MBDs, highlighting their ability to improve pharmacokinetic profiles and therapeutic efficacy. Finally, the review critically examines current hurdles regarding manufacturing, safety, and clinical translation, and offers perspectives on next-generation systems that combine diagnostic sensing with adaptive therapy to realize personalized bone health management.

Objective: To evaluate the efficacy of combined umbilical adipose tissue and venous blood-derived stromal vascular fraction (SVF) on limb perfusion, pain and functional recovery in patients with chronic limb-threatening ischemia (CLTI).

Methods: A total of 20 patients aged between 36 and 82 years (mean age: 60.3 ± 11.2 years) were included in the study. Ankle-Brachial Index (ABI), pain scores and pain-free walking distance were evaluated before treatment and at follow-up at 3, 6, 9 and 12 months.

Results: At 12 months, ABI increased significantly, Visual Analog Scale (VAS) decreased, while pain-free walking distance improved at 9 and 12 months compared to baseline. Left ventricular ejection fraction (LVEF) showed a non-significant rise (54.6%-55.2%, p = 0.072), while renal function improved modestly (creatinine 1.12 → 1.09 mg/dL, eGFR 78 → 80.5 mL/min/1.73 m², p = 0.034). Angiography revealed enhanced vessel visualization and collateral formation.

Conclusions: Combined Autologous conditioned plasma (ACP) and adipose-derived SVF injections effectively improved limb perfusion, pain, lesion severity and functional outcomes in CLTI patients, suggesting a promising therapeutic approach for peripheral arterial disease (PAD).

Movement monitoring with wearable technologies is becoming increasingly popular in different fields of application (clinical, sports, entertainment). Particularly, textile-based wearables for movement monitoring are attractive as they follow the body movement, are comfortable to use, and can provide continuous tracking capabilities. Ideally, these wearable devices should be flexible (as opposed to current technologies with rigid electronics on the garments) and transmit data wirelessly to avoid hindering the natural movement with connections. Although fully textile wireless and passive wearable systems - whereby the textile sensing part does not have any rigid components and the data is wirelessly transmitted to an external reader - have been developed, the capability of these technologies is currently limited to a single sensor. In this work, we present a system based on a resonating inductor-capacitor (LC) circuits that can measure multiple sensors to broaden the range of use by tracking more than a single joint. Importantly, the presented system employs multiple capacitive strain sensors but retains the use of a single inductor for data transmission, limiting the complexity of realization and the number of connections. After characterization on the bench for careful design of the circuit components, we demonstrated the capability of the system to be used for human movement monitoring and activity classification by integrating two sensors in sport leggings and performing different static and dynamic activities. The tests with sensorized leggings were performed by a single participant. Among a set of chosen classification algorithms, the best performance (F1-score) was 0.98 for the static activities and 0.96 for dynamic activities. When including three independent sessions (donning and doffing the sensorised leggings) accuracy and F1-score dropped to 0.86 and 0.87 respectively. Overall, the presented system has the potential to be adopted as unobtrusive and comfortable smart clothing for real time movement monitoring.