Three-Dimensional Extracellular Matrix Protein Hydrogels for Human Trabecular Meshwork Cell Studies.

The extracellular microenvironment plays a crucial role in regulating a wide range of cell behaviors. Biopolymer hydrogels are ideally suited to present a realistic three-dimensional extracellular milieu to cells in vitro. Here, we describe the fabrication and use of soft tissue-mimetic extracellular matrix protein hydrogels for investigations of human trabecular meshwork cell biology.

Navigating the combinations of platelet-rich fibrin with biomaterials used in maxillofacial surgery.

Platelet-rich fibrin (PRF) is a protein matrix with growth factors and immune cells extracted from venous blood via centrifugation. Previous studies proved it a beneficial biomaterial for bone and soft tissue regeneration in dental surgeries. Researchers have combined PRF with a wide range of biomaterials for composite preparation as it is biocompatible and easily acquirable. The results of the studies are difficult to compare due to varied research methods and the fact that researchers focus more on the PRF preparation protocol and less on the interaction of PRF with the chosen material. Here, the literature from 2013 to 2024 is reviewed to help surgeons and researchers navigate the field of commonly used biomaterials in maxillofacial surgeries (calcium phosphate bone grafts, polymers, metal nanoparticles, and novel composites) and their combinations with PRF. The aim is to help the readers select a composite that suits their planned research or medical case. Overall, PRF combined with bone graft materials shows potential for enhancing bone regeneration both in vivo and in vitro. Still, results vary across studies, necessitating standardized protocols and extensive clinical trials. Overviewed methods showed that the biological and mechanical properties of the PRF and material composites can be altered depending on the PRF preparation and incorporation process.

Engineering branched ionizable lipid for hepatic delivery of clustered regularly interspaced short palindromic repeat-Cas9 ribonucleoproteins.

The delivery of the CRISPR/Cas ribonucleoprotein (RNP) has received attention for clinical applications owing to its high efficiency with few off-target effects. Lipid nanoparticles (LNPs) are potential non-viral vectors for the delivery of RNPs. Herein, we report the engineering of a branched scaffold structure of ionizable lipids for the hepatic delivery of RNPs. Both the total carbon number and branching position were critical for the functional delivery of RNPs. The optimal ionizable lipid exhibited a more than 98% reduction in transthyretin protein after a single dose with no obvious signs of toxicity. The mechanistic study has revealed that optimal LNPs have a unique "flower-like structure" that depends on both the lipid structure and the payload and that these LNPs accumulate in hepatocytes in an apolipoprotein E-independent manner. These results represent a major step toward the realization of in vivo genome editing therapy via RNP delivery using chemically synthesizable LNP formulations.

Advances in Tissue Engineering and Its Future in Regenerative Medicine Compared to Traditional Reconstructive Techniques: A Comparative Analysis.

Tissue engineering represents a revolutionary approach in regenerative medicine, offering promising alternatives to traditional reconstructive techniques. This systematic review explores recent advances in tissue engineering, comparing their efficacy, postoperative outcomes, and patient satisfaction to conventional methods. A comprehensive literature search was conducted across PubMed, Cochrane Library, and Google Scholar, covering studies published from 2000 to 2024. Fourteen studies were selected for final analysis based on inclusion criteria focusing on outcomes such as scar quality, postoperative pain, and patient satisfaction. The review demonstrated that tissue engineering techniques consistently provided superior cosmetic outcomes with minimal scarring compared to traditional methods. Patients undergoing tissue-engineered procedures experienced mild-to-moderate postoperative pain with rapid resolution, whereas traditional techniques resulted in moderate to severe pain requiring extended management. Furthermore, patients treated with tissue engineering reported high satisfaction rates due to improved cosmetic and functional outcomes. Despite challenges such as ensuring adequate vascularization, controlling scaffold degradation, and overcoming regulatory and cost barriers, ongoing research and development are essential to fully realize the potential of these innovative therapies. Tissue engineering offers significant advantages over traditional reconstructive techniques and has the potential to profoundly improve patient care in regenerative medicine.

Investigation into the significant role of dermal-epidermal interactions in skin ageing utilising a bioengineered skin construct.

Increased prevalence of skin ageing is a growing concern due to an ageing global population and has both sociological and psychological implications. The use of more clinically predictive in vitro methods for dermatological research is becoming commonplace due to initiatives and the cost of clinical testing. In this study, we utilise a well-defined and characterised bioengineered skin construct as a tool to investigate the cellular and molecular dynamics involved in skin ageing from a dermal perspective. Through incorporation of ageing fibroblasts into the dermal compartment we demonstrate the significant impact of dermal-epidermal crosstalk on the overlying epidermal epithelium. We characterise the paracrine nature of dermal-epidermal communication and the impact this has during skin ageing. Soluble factors, such as inflammatory cytokines released as a consequence of senescence associated secretory phenotype (SASP) from ageing fibroblasts, are known to play a pivotal role in skin ageing. Here, we demonstrate their effect on epidermal morphology and thickness, but not keratinocyte differentiation or tissue structure. Through a novel in vitro strategy utilising bioengineered tissue constructs, this study offers a unique reductionist approach to study epidermal and dermal compartments in isolation and tandem.

The Assembly of Miniaturized Droplets toward Functional Architectures.

Recent explorations of bioengineering have generated new concepts and strategies for the processing of soft and functional materials. Droplet assembly techniques can address problems in the construction of extremely soft architectures by expanding the manufacturing capabilities using droplets containing liquid or hydrogels including weak hydrogels. This Perspective sets out to provide a brief overview of this growing field, and discusses the challenges and opportunities ahead. The study highlights the recent key advances of materials and architectures from hitherto effective droplet-assembly technologies, as well as the applications in biomedical and bioengineering fields from artificial tissues to bioreactors. It is envisaged that these assembled architectures, as nature-inspired models, will stimulate the discovery of biomaterials and miniaturized platforms for interdisciplinary research in health, biotechnology, and sustainability.

Exploring the use of deep learning models for accurate tracking of 3D zebrafish trajectories.

Zebrafish are ideal model organisms for various fields of biological research, including genetics, neural transmission patterns, disease and drug testing, and heart disease studies, because of their unique ability to regenerate cardiac muscle. Tracking zebrafish trajectories is essential for understanding their behavior, physiological states, and disease associations. While 2D tracking methods are limited, 3D tracking provides more accurate descriptions of their movements, leading to a comprehensive understanding of their behavior. In this study, we used deep learning models to track the 3D movements of zebrafish. Videos were captured by two custom-made cameras, and 21,360 images were labeled for the dataset. The YOLOv7 model was trained using hyperparameter tuning, with the top- and side-view camera models trained using the v7x.pt and v7.pt weights, respectively, over 300 iterations with 10,680 data points each. The models achieved impressive results, with an accuracy of 98.7% and a recall of 98.1% based on the test set. The collected data were also used to generate dynamic 3D trajectories. Based on a test set with 3,632 3D coordinates, the final model detected 173.11% more coordinates than the initial model. Compared to the ground truth, the maximum and minimum errors decreased by 97.39% and 86.36%, respectively, and the average error decreased by 90.5%.This study presents a feasible 3D tracking method for zebrafish trajectories. The results can be used for further analysis of movement-related behavioral data, contributing to experimental research utilizing zebrafish.

Improving tumor microenvironment assessment in chip systems through next-generation technology integration.

The tumor microenvironment (TME) comprises a diverse array of cells, both cancerous and non-cancerous, including stromal cells and immune cells. Complex interactions among these cells play a central role in driving cancer progression, impacting critical aspects such as tumor initiation, growth, invasion, response to therapy, and the development of drug resistance. While targeting the TME has emerged as a promising therapeutic strategy, there is a critical need for innovative approaches that accurately replicate its complex cellular and non-cellular interactions; the goal being to develop targeted, personalized therapies that can effectively elicit anti-cancer responses in patients. Microfluidic systems present notable advantages over conventional in vitro 2D co-culture models and in vivo animal models, as they more accurately mimic crucial features of the TME and enable precise, controlled examination of the dynamic interactions among multiple human cell types at any time point. Combining these models with next-generation technologies, such as bioprinting, single cell sequencing and real-time biosensing, is a crucial next step in the advancement of microfluidic models. This review aims to emphasize the importance of this integrated approach to further our understanding of the TME by showcasing current microfluidic model systems that integrate next-generation technologies to dissect cellular intra-tumoral interactions across different tumor types. Carefully unraveling the complexity of the TME by leveraging next generation technologies will be pivotal for developing targeted therapies that can effectively enhance robust anti-tumoral responses in patients and address the limitations of current treatment modalities.

Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation.

Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.

A Bioengineered Cathepsin B-sensitive Gas Vesicle Nanosystem That Responds With Increased Gray-level Intensity of Ultrasound Biomicroscopic Images.

OBJECTIVE: This work aimed to promote the interaction of a modified gas vesicle (GV) with cathepsin B (CTSB) protease and analysed their backscattered signal by ultrasound (US). METHODS: We modified the sequence of the gene coding for GvpC to contain a CTSB cleavage and expressed the protein in an Escherichia coli recombinant system. The protein was purified and added to GVs preparations in which the original GvpC was removed (ΔGV), constituting the modified GV (GV*). Western blot testing was used to compare GVs with GvpC and engineered GvpC at starting (T0) and after 24 h (T24) reacting with CTSB. A 21 MHz US B-mode and non-linear contrast mode (5% total power) imaged US phantoms having samples of GVwt, ΔGV (stripped GV), GV* and CTSB + GV*. Also, a 21 MHz US B-mode imaged US phantoms having a tumour cell line extracellular fraction (TCEF) and the TCEF + GV* sample. A 100% total US power was applied to collapse the GV structure. RESULTS: On Western blotting, we detected a decrease in engineered GvpC levels 24 h after the incubation of GV* with CTSB, compared with the concentration at T0, suggesting that CTSB cleaved the engineered GvpC. Regions-of-interest over image of phantom cross-sections were determined and the B-mode image mean grey-level intensity resulted in a significant (p < 0.05) increase comparing CTSB + GV* with PBS (control), GVwt, ΔGV and GV*. Non-linear mode image grey-level intensity from CTSB + GV* increased by 11.79, 7.86 and 14.75 dB from samples containing GVwt, ΔGV and GV*, respectively. GV preparations incubated with TCEF and the TCEF + GV* sample showed an increase of 81% in signal compared with TCEF + GVwt. CONCLUSION: The increased US backscattered signal intensity suggests GVs as a potential biosensor for protease activity, possibly aiding the detection of protease-rich tissue regions.

Protocol to investigate G protein-coupled receptor signaling kinetics and concentration-dependent responses using ONE-GO biosensors.

ONE vector G protein optical (ONE-GO) biosensors are versatile tools to measure the activity of G protein-coupled receptors (GPCRs) in cells. The availability of ONE-GO biosensors for ten active Gα subunits representative of all four G protein families (Gs, Gi/o, Gq/11, and G12/13) permits the study of virtually any GPCR. Here, we present a protocol to implement ONE-GO biosensors in cell lines to investigate GPCR signaling kinetics and concentration-dependent responses. We describe steps for cell culture and transfection, response measurement, and data analysis. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.

Exploring immune response toward transplanted human kidney tissues assembled from organoid building blocks.

The increasing scarcity of organs and the significant morbidity linked to dialysis require the development of engineered kidney tissues from human-induced pluripotent stem cells. Integrative approaches that synergize scalable kidney organoid differentiation, tissue biomanufacturing, and comprehensive assessment of their immune response and host integration are essential to accomplish this. Here, we create engineered human kidney tissues composed of organoid building blocks (OBBs) and transplant them into mice reconstituted with allogeneic human immune cells. Tissue-infiltrating human immune cells are composed of effector T cells and innate cells. This immune infiltration leads to kidney tissue injury characterized by reduced microvasculature, enhanced kidney cell apoptosis, and an inflammatory gene signature comparable to kidney organ transplant rejection in humans. Upon treatment with the immunosuppressive agent rapamycin, the induced immune response is greatly suppressed. Our model is a translational platform to study engineered kidney tissue immunogenicity and develop therapeutic targets for kidney rejection.

FSTL-1 loaded 3D bioprinted vascular patch regenerates the ischemic heart tissue.

Cardiac patch strategies are developed as a promising approach to regenerate the injured heart after myocardial infarction (MI). This study integrated 3D bioprinting and cardioprotective paracrine signaling to fabricate vascular patch devices containing endothelial cells (ECs) and the regenerative follistatin-like 1 (FSTL1) peptide. Engineered patch supported the 3D culture of ECs in both static and dynamic culture, forming a uniform endothelium on the printed channels. Implantation of vascular patch onto a rat model of acute MI resulted in significant reduction of scar formation, left ventricle dilation, and wall thinning, as well as enhanced ejection fraction. Furthermore, increased vascularization and proliferation of cardiomyocytes were observed in hearts treated with patches. These findings highlight the remarkable capacity of 3D bioprinted vascular patch to augment the endogenous regenerative capacity of mammalian heart, together with the exogenous cardioprotective function, to serve as a robust therapeutic device to treat acute MI.

Protocol for 3D bioprinting of nanoparticle-laden hydrogels to enhance antibacterial and imaging properties.

This protocol describes the preparation of a nanoparticle-encapsulated bioink capable of protecting tissue-engineered constructs against bacterial infections while also providing contrast for magnetic resonance (MR) imaging modalities. The report includes details of preparing the methacrylated gelatin-based bioinks and the incorporation of superparamagnetic iron oxide nanoparticles. A detailed protocol is presented for characterizing the bioink, evaluating cell response, and assessing its antibacterial effect. Overall, this article presents a robust approach for the development of antibacterial, MR-visible bioinks suitable for various tissue engineering applications. For complete details on the use and execution of this protocol, please refer to Theus et al.1.

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (Gs, Gi/o, Gq/11, G12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.

Recent research progresses of bioengineered biliary stents.

Bile duct lesion, including benign (eg. occlusion, cholelithiasis, dilatation, malformation) and malignant (cholangiocarcinoma) diseases, is a frequently encountered challenge in hepatobiliary diseases, which can be repaired by interventional or surgical procedures. A viable cure for bile duct lesions is implantation with biliary stents. Despite the placement achieved by current clinical biliary stents, the creation of functional and readily transplantable biliary stents remains a formidable obstacle. Excellent biocompatibility, stable mechanics, and absorbability are just a few benefits of using bioengineered biliary stents, which can also support and repair damaged bile ducts that drain bile. Additionally, cell sources & organoids derived from the biliary system that are loaded onto scaffolds can encourage bile duct regeneration. Therefore, the implantation of bioengineered biliary stent is considered as an ideal treatment for bile duct lesion, holding a broad potential for clinical applications in future. In this review, we look back on the development of conventional biliary stents, biodegradable biliary stents, and bioengineered biliary stents, highlighting the crucial elements of bioengineered biliary stents in promoting bile duct regeneration. After providing an overview of the various types of cell sources & organoids and fabrication methods utilized for the bioengineering process, we present the in vitro and in vivo applications of bioengineered biliary ducts, along with the latest advances in this exciting field. Finally, we also emphasize the ongoing challenges and future development of bioengineered biliary stents.

Innervation in corneal bioengineering.

Given the crucial role nerves play in maintaining corneal function and integrity, the ability of bioengineered cornea to demonstrate functional nerve regeneration directly influences their longevity and stability. Despite advances in biofabrication techniques and an increasing appreciation of the importance of neural innervation, to this day none have completely replicated the complexity and functionality of the cornea with successful innervation. This review evaluates the materials and fabrication techniques used to produce and enhance innervation in bioengineered cornea. Approaches to facilitating innervation are discussed and methods of assessing innervation compared. Finally, current challenges and future directions for innervated bioengineered cornea are presented, providing guidance for future work. STATEMENT OF SIGNIFICANCE: The functional nerve regeneration in bioengineered corneas directly influences their longevity and stability. Despite advancements in biofabrication techniques and growing recognition of the importance of neural innervation for bioengineered cornea, there remains a lack of comprehensive reviews on this topic. This review addresses the critical gap by evaluating the materials and fabrication techniques employed to promote innervation in bioengineered corneas. Additionally, we discuss various approaches to enhancing innervation, compare assessment methods, and examine both in vitro and in vivo responses. By providing a comprehensive overview of the current state of research and highlighting challenges and future directions, this review aims to provide guidance for inducing innervation of bioengineered cornea.

Analysis of the positive expiratory pressure valves of coupled oronasal mask.

Positive expiratory pressure (PEP) is a technique used in respiratory physiotherapy to treat diseases related to the respiratory system through spontaneous breathing. This equipment consists of an oronasal mask coupled to a T connector with a unidirectional valve. Studies that evaluate whether the pressure level in the one-way valve corresponds to the actual pressure level provided are scarce in the scientific literature. In order to investigate the failures, bench tests were carried out on the spring-loaded valves, using a U-tube manometer. This pressure was exerted on the valve using a syringe that generated air flow inside the U-tube, allowing analysis numerical value of the measured pressure and the specified values of the valve, thus verifying the disparity of these measured values in relation to the PEP values operated by valves (0 to 20 cmH2O) from the three manufacturers under study. PEPs generated by spring-loaded valves from all three manufacturers were higher than pressures in the range of 2.5 to 20 cmH2O, with significant differences between manufacturers. This bench study showed inaccurate operation of all spring-loaded PEP valves of the three manufactures. The results obtained and the performance of the valves require a reevaluation of manufacturing procedures to preserve product quality and efficacy in clinical application.

In vivo thrombin activity in the diatom Phaeodactylum tricornutum: biotechnological insights.

Diatoms are responsible for 20% of global carbon dioxide fixation and have significant potential in various biotechnological and industrial applications. Recently, the pennate diatom Phaeodactylum tricornutum has emerged as a prominent platform organism for metabolic engineering and synthetic biology. The availability of its genome sequence has facilitated the development of new bioengineering tools. In this study, we used in silico analyses to identify sequences potentially encoding thrombin-like proteins, which are involved in recognizing and cleaving the thrombin sequence LVPRGS in P. tricornutum. Protein structure prediction and docking studies indicated a similar active site and ligand positioning compared to characterized human and bovine thrombin. The evidence and efficiency of the cleavage were determined in vivo using two fusion-protein constructs that included YFP to measure expression, protein accumulation, and cleavage. Western blot analysis revealed 50-100% cleavage between YFP and N-terminal fusion proteins. Our findings suggest the existence of a novel thrombin-like protease in P. tricornutum. This study advances the application of diatoms for the synthesis and production of complex proteins and enhances our understanding of the functional role of these putative thrombin sequences in diatom physiology. KEY POINTS: • Protein structure predictions reveal thrombin-like active sites in P. tricornutum. • Validated cleavage efficiency of thrombin-like protease on fusion proteins in vivo. • Study advances bioengineering tools for diatom-based biotechnological applications.

Molecular-based characterization and bioengineering of Sorghum bicolor to enhance iron deficiency tolerance in iron-limiting calcareous soils.

Plant biomass can significantly contribute to alternative energy sources. Sorghum bicolor is a promising plant for producing energy, but is susceptible to iron deficiency, which inhibits its cultivation in iron-limiting calcareous soils. The molecular basis for the susceptibility of sorghum to iron deficiency remains unclear. Here, we explored the sorghum genome to identify genes involved in iron uptake and translocation. Iron deficiency-responsive gene expression was comparable to that in other graminaceous plants. A nicotianamine synthase gene, SbNAS1, was induced in response to iron deficiency, and SbNAS1 showed enzyme activity. Sorghum secreted 2'-deoxymugineic acid and other phytosiderophores under iron deficiency, but their levels were relatively low. Intercropping of sorghum with barley or rice rescued iron deficiency symptoms of sorghum. To produce bioengineered sorghum with enhanced tolerance to iron deficiency, we introduced four cassettes into sorghum: 35S promoter-OsIRO2 for activation of iron acquisition-related gene expression, SbIRT1 promoter-Refre1/372 for enhanced ferric-chelate reductase activity, and barley IDS3, and HvNAS1 genomic fragments for enhanced production of phytosiderophores and nicotianamine. The resultant single sorghum line exhibited enhanced secretion of phytosiderophores, increased ferric-chelate reductase activity, and improved iron uptake and leaf greenness compared with non-transformants under iron-limiting conditions. Similar traits were also conferred to rice by introducing the four cassettes. Moreover, these rice lines showed similar or better tolerance in calcareous soils and increased grain iron accumulation compared with previous rice lines carrying two or three comparable cassettes. These results provide a molecular basis for the bioengineering of sorghum tolerant of low iron availability in calcareous soils.