Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

Bioengineering Skin Substitutes for Wound Management-Perspectives and Challenges.

Non-healing wounds and skin losses constitute significant challenges for modern medicine and pharmacology. Conventional methods of wound treatment are effective in basic healthcare; however, they are insufficient in managing chronic wound and large skin defects, so novel, alternative methods of therapy are sought. Among the potentially innovative procedures, the use of skin substitutes may be a promising therapeutic method. Skin substitutes are a heterogeneous group of materials that are used to heal and close wounds and temporarily or permanently fulfill the functions of the skin. Classification can be based on the structure or type (biological and synthetic). Simple constructs (class I) have been widely researched over the years, and can be used in burns and ulcers. More complex substitutes (class II and III) are still studied, but these may be utilized in patients with deep skin defects. In addition, 3D bioprinting is a rapidly developing method used to create advanced skin constructs and their appendages. The aforementioned therapies represent an opportunity for treating patients with diabetic foot ulcers or deep skin burns. Despite these significant developments, further clinical trials are needed to allow the use skin substitutes in the personalized treatment of chronic wounds.

Controllable and reusable seesaw circuit based on nicking endonucleases.

Seesaw circuits are essential for molecular computing and biosensing. However, a notable limitation of seesaw circuits lies in the irreversible depletion of components, precluding the attainment of system recovery and rendering nucleic acid circuits non-reusable. We developed a brand-new method for creating controllable and reusable seesaw circuits. By using the nicking endonucleases Nt.BbvCI and Nt.Alwi, we removed "functional components" while keeping the "skeletal components" for recurrent usage. T-inputs were introduced, increasing the signal-to-noise ratio of AND logic from 2.68 to 11.33 and demonstrating compatibility. We identified the logic switching feature and verified that it does not impair circuit performance. We also built intricate logic circuits, such as OR-AND gate, to demonstrate the versatility of our methodology. This controllable reusability extends the applications of nanotechnology and bioengineering, enhancing the practicality and efficiency of these circuits across various domains.

Engineering is evolution: a perspective on design processes to engineer biology.

Careful consideration of how we approach design is crucial to all areas of biotechnology. However, choosing or developing an effective design methodology is not always easy as biology, unlike most areas of engineering, is able to adapt and evolve. Here, we put forward that design and evolution follow a similar cyclic process and therefore all design methods, including traditional design, directed evolution, and even random trial and error, exist within an evolutionary design spectrum. This contrasts with conventional views that often place these methods at odds and provides a valuable framework for unifying engineering approaches for challenging biological design problems.

Iterative nanoparticle bioengineering enabled by x-ray fluorescence imaging.

Nanoparticles (NPs) are currently developed for drug delivery and molecular imaging. However, they often get intercepted before reaching their target, leading to low targeting efficacy and signal-to-noise ratio. They tend to accumulate in organs like lungs, liver, kidneys, and spleen. The remedy is to iteratively engineer NP surface properties and administration strategies, presently a time-consuming process that includes organ dissection at different time points. To improve this, we propose a rapid iterative approach using whole-animal x-ray fluorescence (XRF) imaging to systematically evaluate NP distribution in vivo. We applied this method to molybdenum-based NPs and clodronate liposomes for tumor targeting with transient macrophage depletion, leading to reduced accumulations in lungs and liver and eventual tumor detection. XRF computed tomography (XFCT) provided 3D insight into NP distribution within the tumor. We validated the results using a multiscale imaging approach with dye-doped NPs and gene expression analysis for nanotoxicological profiling. XRF imaging holds potential for advancing therapeutics and diagnostics in preclinical pharmacokinetic studies.

teemi: An open-source literate programming approach for iterative design-build-test-learn cycles in bioengineering.

Synthetic biology dictates the data-driven engineering of biocatalysis, cellular functions, and organism behavior. Integral to synthetic biology is the aspiration to efficiently find, access, interoperate, and reuse high-quality data on genotype-phenotype relationships of native and engineered biosystems under FAIR principles, and from this facilitate forward-engineering strategies. However, biology is complex at the regulatory level, and noisy at the operational level, thus necessitating systematic and diligent data handling at all levels of the design, build, and test phases in order to maximize learning in the iterative design-build-test-learn engineering cycle. To enable user-friendly simulation, organization, and guidance for the engineering of biosystems, we have developed an open-source python-based computer-aided design and analysis platform operating under a literate programming user-interface hosted on Github. The platform is called teemi and is fully compliant with FAIR principles. In this study we apply teemi for i) designing and simulating bioengineering, ii) integrating and analyzing multivariate datasets, and iii) machine-learning for predictive engineering of metabolic pathway designs for production of a key precursor to medicinal alkaloids in yeast. The teemi platform is publicly available at PyPi and GitHub.

Bioengineering Full-scale auricles using 3D-printed external scaffolds and decellularized cartilage xenograft.

Reconstruction of the human auricle remains a formidable challenge for plastic surgeons. Autologous costal cartilage grafts and alloplastic implants are technically challenging, and aesthetic and/or tactile outcomes are frequently suboptimal. Using a small animal "bioreactor", we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimics the size, shape, and biomechanical properties of the native human auricle. The full-scale polylactic acid ear scaffolds were 3D-printed based upon data acquired from 3D photogrammetry of an adult ear. Ovine costal cartilage was processed either through mincing (1 mm3) or zesting (< 0.5 mm3), and then fully decellularized and sterilized. At explantation, both the minced and zested neoears maintained the size and contour complexities of the scaffold topography with steady tissue ingrowth through 6 months in vivo. A mild inflammatory infiltrate at 3 months was replaced by homogenous fibrovascular tissue ingrowth enveloping individual cartilage pieces at 6 months. All ear constructs were pliable, and the elasticity was confirmed by biomechanical analysis. Longer-term studies of the neoears with faster degrading biomaterials will be warranted for future clinical application. STATEMENT OF SIGNIFICANCE: Accurate reconstruction of the human auricle has always been a formidable challenge to plastic surgeons. In this article, we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimic the size, shape, and biomechanical properties of the native human auricle. Longer-term studies of the neoears with faster degrading biomaterials will be warranted for future clinical application.

Collective intelligence: A unifying concept for integrating biology across scales and substrates.

A defining feature of biology is the use of a multiscale architecture, ranging from molecular networks to cells, tissues, organs, whole bodies, and swarms. Crucially however, biology is not only nested structurally, but also functionally: each level is able to solve problems in distinct problem spaces, such as physiological, morphological, and behavioral state space. Percolating adaptive functionality from one level of competent subunits to a higher functional level of organization requires collective dynamics: multiple components must work together to achieve specific outcomes. Here we overview a number of biological examples at different scales which highlight the ability of cellular material to make decisions that implement cooperation toward specific homeodynamic endpoints, and implement collective intelligence by solving problems at the cell, tissue, and whole-organism levels. We explore the hypothesis that collective intelligence is not only the province of groups of animals, and that an important symmetry exists between the behavioral science of swarms and the competencies of cells and other biological systems at different scales. We then briefly outline the implications of this approach, and the possible impact of tools from the field of diverse intelligence for regenerative medicine and synthetic bioengineering.

Bioengineering toolkits for potentiating organoid therapeutics.

Organoids are three-dimensional, multicellular constructs that recapitulate the structural and functional features of specific organs. Because of these characteristics, organoids have been widely applied in biomedical research in recent decades. Remarkable advancements in organoid technology have positioned them as promising candidates for regenerative medicine. However, current organoids still have limitations, such as the absence of internal vasculature, limited functionality, and a small size that is not commensurate with that of actual organs. These limitations hinder their survival and regenerative effects after transplantation. Another significant concern is the reliance on mouse tumor-derived matrix in organoid culture, which is unsuitable for clinical translation due to its tumor origin and safety issues. Therefore, our aim is to describe engineering strategies and alternative biocompatible materials that can facilitate the practical applications of organoids in regenerative medicine. Furthermore, we highlight meaningful progress in organoid transplantation, with a particular emphasis on the functional restoration of various organs.

The promising prospect of human hair follicle regeneration in the shadow of new tissue engineering strategies.

Hair loss disorder (alopecia) affects numerous people around the world. The low effectiveness and numerous side effects of common treatments have prompted researchers to investigate alternative and effective solutions. Hair follicle (HF) bioengineering is the knowledge of using hair-inductive (trichogenic) cells. Most bioengineering-based approaches focus on regenerating folliculogenesis through manipulation of regulators of physical/molecular properties in the HF niche. Despite the high potential of cell therapy, no cell product has been produced for effective treatment in the field of hair regeneration. This problem shows the challenges in the functionality of cultured human hair cells. To achieve this goal, research and development of new and practical approaches, technologies and biomaterials are needed. Based on recent advances in the field, this review evaluates emerging HF bioengineering strategies and the future prospects for the field of tissue engineering and successful HF regeneration.

Advances in microbial exoenzymes bioengineering for improvement of bioplastics degradation.

Plastic pollution has become a major global concern, posing numerous challenges for the environment and wildlife. Most conventional ways of plastics degradation are inefficient and cause great damage to ecosystems. The development of biodegradable plastics offers a promising solution for waste management. These plastics are designed to break down under various conditions, opening up new possibilities to mitigate the negative impact of traditional plastics. Microbes, including bacteria and fungi, play a crucial role in the degradation of bioplastics by producing and secreting extracellular enzymes, such as cutinase, lipases, and proteases. However, these microbial enzymes are sensitive to extreme environmental conditions, such as temperature and acidity, affecting their functions and stability. To address these challenges, scientists have employed protein engineering and immobilization techniques to enhance enzyme stability and predict protein structures. Strategies such as improving enzyme and substrate interaction, increasing enzyme thermostability, reinforcing the bonding between the active site of the enzyme and substrate, and refining enzyme activity are being utilized to boost enzyme immobilization and functionality. Recently, bioengineering through gene cloning and expression in potential microorganisms, has revolutionized the biodegradation of bioplastics. This review aimed to discuss the most recent protein engineering strategies for modifying bioplastic-degrading enzymes in terms of stability and functionality, including enzyme thermostability enhancement, reinforcing the substrate binding to the enzyme active site, refining with other enzymes, and improvement of enzyme surface and substrate action. Additionally, discovered bioplastic-degrading exoenzymes by metagenomics techniques were emphasized.

Crop bioengineering via gene editing: reshaping the future of agriculture.

Genome-editing technologies have revolutionized research in plant biology, with major implications for agriculture and worldwide food security, particularly in the face of challenges such as climate change and increasing human populations. Among these technologies, clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated protein [Cas] systems are now widely used for editing crop plant genomes. In this review, we provide an overview of CRISPR-Cas technology and its most significant applications for improving crop sustainability. We also review current and potential technological advances that will aid in the future breeding of crops to enhance food security worldwide. Finally, we discuss the obstacles and challenges that must be overcome to realize the maximum potential of genome-editing technologies for future crop and food production.

Transplantation of a bioengineered tissue patch promotes uterine repair in the sheep.

Innovative bioengineering strategies utilizing extracellular matrix (ECM) based scaffolds derived from decellularized tissue offer new prospects for restoring damaged uterine tissue. Despite successful fertility restoration in small animal models, the translation to larger and more clinically relevant models have not yet been assessed. Thus, our study investigated the feasibility to use a 6 cm2 graft constructed from decellularized sheep uterine tissue, mimicking a future application to repair a uterine defect in women. Some grafts were also recellularized with fetal sheep bone marrow-derived mesenchymal stem cells (SF-MSCs). The animals were followed for six weeks post-surgery during which blood samples were collected to assess the systemic immune cell activation by fluorescence-activated cell sorting (FACS) analysis. Tissue regeneration was assessed by histology, immunohistochemistry, and gene expression analyses. There was a large intra-group variance which prompted us to implement a novel scoring system to comprehensively evaluate the regenerative outcomes. Based on the regenerative score each graft received, we focused our analysis to map potential differences that may have played a role in the success or failure of tissue repair following the transplantation therapy. Notably, three out of 15 grafts exhibited major regeneration that resembled native uterine tissue, and an additional three grafts showed substantial regenerative outcomes. For the better regenerated grafts, it was observed that the systemic T-cell subgroups were significantly different compared with the failing grafts. Hence, our data suggest that the T-cell response play an important role for determining the uterus tissue regeneration outcomes. The remarkable regeneration seen in the best-performing grafts after just six weeks following transplantation provides compelling evidence that decellularized tissue for uterine bioengineering holds great promise for clinically relevant applications.

Bioengineering of vascularized porcine flaps using perfusion-recellularization.

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Thermophilic ß-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

ß-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable ß-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic ß-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

[Integration of "tri-bio, tri-chain and tri-creation" innovation and entrepreneurship education into talent training program].

Under the background of the "era of mass innovation", there are challenges in the training of biotechnology professionals, including a "backward concept of innovation and entrepreneurship education", a "singular education method of innovation and entrepreneurship", and a "limited practice platform of innovation and entrepreneurship". These challenges require the implementation of a new training model. In comparison to the talent training objectives of new engineering construction, the College of Biotechnology and Bioengineering at Zhejiang University of Technology has been exploring and practicing the training mode "tri-bio, tri-chain and tri-creation " for 42 years. The research has established a new platform and paradigm for training exceptional engineering innovation and entrepreneurship talents. It also offers valuable references and insights for the reform of training methods for biotechnology professionals by optimizing the education concept of "biology, life and live ", enriching the education method of "knowledge chain, scientific research chain and industrial chain", and building the three-creation technology practice platform based on "creativity, innovation and entrepreneurship".