Biofabrication最新文献

The fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here, we implemented a 3D model of cardiac vasculogenesis, incorporating endothelial cells (EC), stromal cells, and human iPSC-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues, resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis, including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs, the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast, supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly, enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization, illustrating the use of this approach in the engineering of enhanced, perfusable, microfluidic models of the myocardium.

The viscosity of gelatin methacryloyl (GelMA)-based bioinks generates shear stresses throughout the printing process that can affect cell integrity, reduce cell viability, cause morphological changes, and alter cell functionality. This study systematically investigated the impact of the viscosity of GelMA-gelatin bioinks on osteoblast-like cells in 2D and 3D culture conditions. Three bioinks with low, medium, and high viscosity prepared by supplementing a 5% GelMA solution with different concentrations of gelatin were evaluated. Cell responses were studied in a 2D environment after printing and incubation in non-cross-linked bioinks that caused the gelatin and GelMA to dissolve and release cells for attachment to tissue culture plates. The increased viscosity of the bioinks significantly affected cell area and aspect ratio. Cells printed using the bioink with medium viscosity exhibited greater metabolic activity and proliferation rate than those printed using the high viscosity bioink and even the unprinted control cells. Additionally, cells printed using the bioink with high viscosity demonstrated notably elevated expression levels of alkaline phosphatase and bone morphogenetic protein-2 genes. In the 3D condition, the printed cell-laden hydrogels were photo-cross-linked prior to incubation. The medium viscosity bioink supported greater cell proliferation compared to the high viscosity bioink. However, there were no significant differences in the expression of osteogenic markers between the medium and high viscosity bioinks. Therefore, the choice between medium and high viscosity bioinks should be based on the desired outcomes and objectives of the bone tissue engineering application. Furthermore, the bioprinting procedure with the medium viscosity bioink was used as an automated technique for efficiently seeding cells onto 3D printed porous titanium scaffolds for bone tissue engineering purposes.

Extracellular vesicles (EVs) show promise in drug loading and delivery for medical applications. However, the lack of scalable manufacturing processes hinders the generation of clinically suitable quantities, thereby impeding the translation of EV-based therapies. Current EV production relies heavily on non-physiological two-dimensional (2D) cell culture or bioreactors, requiring significant resources. Additionally, EV-derived ribonucleic acid cargo in three-dimensional (3D) and 2D culture environments remains largely unknown. In this study, we optimized the biofabrication of 3D auxetic scaffolds encapsulated with human embryonic kidney 293 T (HEK293 T) cells, focusing on enhancing the mechanical properties of the scaffolds to significantly boost EV production through tensile stimulation in bioreactors. The proposed platform increased EV yields approximately 115-fold compared to conventional 2D culture, possessing properties that inhibit tumor progression. Further mechanistic examinations revealed that this effect was mediated by the mechanosensitivity of YAP/TAZ. EVs derived from tensile-stimulated HEK293 T cells on 3D auxetic scaffolds demonstrated superior capability for loading doxorubicin compared to their 2D counterparts for cancer therapy. Our results underscore the potential of this strategy for scaling up EV production and optimizing functional performance for clinical translation.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, a leading cause of cancer-related deaths globally. Initial lesions of PDAC develop within the exocrine pancreas' functional units, with tumor progression driven by interactions between PDAC and stromal cells. Effective therapies require anatomically and functionally relevantin vitrohuman models of the pancreatic cancer microenvironment. We employed tomographic volumetric bioprinting, a novel biofabrication method, to create human fibroblast-laden constructs mimicking the tubuloacinar structures of the exocrine pancreas. Human pancreatic ductal epithelial (HPDE) cells overexpressing the KRAS oncogene (HPDE-KRAS) were seeded in the multiacinar cavity to replicate pathological tissue. HPDE cell growth and organization within the structure were assessed, demonstrating the formation of a thin epithelium covering the acini inner surfaces. Immunofluorescence assays showed significantly higher alpha smooth muscle actin (α-SMA) vs. F-actin expression in fibroblasts co-cultured with cancerous versus wild-type HPDE cells. Additionally,α-SMA expression increased over time and was higher in fibroblasts closer to HPDE cells. Elevated interleukin (IL)-6 levels were quantified in supernatants from co-cultures of stromal and HPDE-KRAS cells. These findings align with inflamed tumor-associated myofibroblast behavior, serving as relevant biomarkers to monitor early disease progression and target drug efficacy. To our knowledge, this is the first demonstration of a 3D bioprinted model of exocrine pancreas that recapitulates its true 3-dimensional microanatomy and shows tumor triggered inflammation.

This study explores the bioprinting of a smooth muscle cell-only bioink into ionically crosslinked oxidized methacrylated alginate (OMA) microgel baths to create self-supporting vascular tissues. The impact of OMA microgel support bath methacrylation degree and cell-only bioink dispensing parameters on tissue formation, remodeling, structure and strength was investigated. We hypothesized that reducing dispensing tip diameter from 27 G (210μm) to 30 G (159μm) for cell-only bioink dispensing would reduce tissue wall thickness and improve the consistency of tissue dimensions while maintaining cell viability. Printing with 30 G tips resulted in decreased mean wall thickness (318.6μm) without compromising mean cell viability (94.8%). Histological analysis of cell-only smooth muscle tissues cultured for 14 d in OMA support baths exhibited decreased wall thickness using 30 G dispensing tips, which correlated with increased collagen deposition and alignment. In addition, a TUNEL assay indicated a decrease in cell death in tissues printed with thinner (30 G) dispensing tips. Mechanical testing demonstrated that tissues printed with a 30 G dispensing tip exhibit an increase in ultimate tensile strength compared to those printed with a 27 G dispensing tip. Overall, these findings highlight the importance of precise control over bioprinting parameters to generate mechanically robust tissues when using cell-only bioinks dispensed and cultured within hydrogel support baths. The ability to control print dimensions using cell-only bioinks may enable bioprinting of more complex soft tissue geometries to generatein vitrotissue models.

Antimicrobial resistance (AMR) poses an emergent threat to global health due to antibiotic abuse, overuse and misuse, necessitating urgent innovative and sustainable solutions. The utilization of bio-nanomaterials as antibiotic allies is a green, economic, sustainable and renewable strategy to combat this pressing issue. These biomaterials involve green precursors (e.g. biowaste, plant extracts, essential oil, microbes, and agricultural residue) and techniques for their fabrication, which reduce their cyto/environmental toxicity and exhibit economic manufacturing, enabling a waste-to-wealth circular economy module. Their nanoscale dimensions with augmented biocompatibility characterize bio-nanomaterials and offer distinctive advantages in addressing AMR. Their ability to target pathogens, such as bacteria and viruses, at the molecular level, coupled with their diverse functionalities and bio-functionality doping from natural precursors, allows for a multifaceted approach to combat resistance. Furthermore, bio-nanomaterials can be tailored to enhance the efficacy of existing antimicrobial agents or deliver novel therapies, presenting a versatile platform for innovation. Their use in combination with traditional antibiotics can mitigate resistance mechanisms, prolong the effectiveness of existing treatments, and reduce side effects. This review aims to shed light on the potential of bio-nanomaterials in countering AMR, related mechanisms, and their applications in various domains. These roles encompass co-therapy, nanoencapsulation, and antimicrobial stewardship, each offering a distinct avenue for overcoming AMR. Besides, it addresses the challenges associated with bio-nanomaterials, emphasizing the importance of regulatory considerations. These green biomaterials are the near future of One Health Care, which will have economic, non-polluting, non-toxic, anti-resistant, biocompatible, degradable, and repurposable avenues, contributing to sustainable development goals.

The importance of hydrogels in tissue engineering cannot be overemphasized due to their resemblance to the native extracellular matrix. However, natural hydrogels with satisfactory biocompatibility exhibit poor mechanical behavior, which hampers their application in stress-bearing soft tissue engineering. Here, we describe the fabrication of a double methacrylated gelatin bioink covalently linked to graphene oxide (GO) via a zero-length crosslinker, digitally light-processed (DLP) printable into 3D complex structures with high fidelity. The resultant natural hydrogel (GelGOMA) exhibits a conductivity of 15.0 S m^-1as a result of the delocalization of theπ-orbital from the covalently linked GO. Furthermore, the hydrogel shows a compressive strength of 1.6 MPa, and a 2.0 mm thick GelGOMA can withstand a 1.0 kg ms^-1momentum. The printability and mechanical strengths of GelGOMAs were demonstrated by printing a fish heart with a functional fluid pumping mechanism and tricuspid valves. Its biocompatibility, electroconductivity, and physiological relevance enhanced the proliferation and differentiation of myoblasts and neuroblasts and the contraction of human-induced pluripotent stem cell-derived cardiomyocytes. GelGOMA demonstrates the potential for the tissue engineering of functional hearts and wearable electronic devices.

Current research practice for optimizing bioink involves exhaustive experimentation with multi-material composition for determining the printability, shape fidelity and biocompatibility. Predicting bioink properties can be beneficial to the research community but is a challenging task due to the non-Newtonian behavior in complex composition. Existing models such as Cross model become inadequate for predicting the viscosity for heterogeneous composition of bioinks. In this paper, we utilize a machine learning framework to accurately predict the viscosity of heterogeneous bioink compositions, aiming to enhance extrusion-based bioprinting techniques. Utilizing Bayesian optimization (BO), our strategy leverages a limited dataset to inform our model. This is a technique especially useful of the typically sparse data in this domain. Moreover, we have also developed a mask technique that can handle complex constraints, informed by domain expertise, to define the feasible parameter space for the components of the bioink and their interactions. Our proposed method is focused on predicting the intrinsic factor (e.g. viscosity) of the bioink precursor which is tied to the extrinsic property (e.g. cell viability) through the mask function. Through the optimization of the hyperparameter, we strike a balance between exploration of new possibilities and exploitation of known data, a balance crucial for refining our acquisition function. This function then guides the selection of subsequent sampling points within the defined viable space and the process continues until convergence is achieved, indicating that the model has sufficiently explored the parameter space and identified the optimal or near-optimal solutions. Employing this AI-guided BO framework, we have developed, tested, and validated a surrogate model for determining the viscosity of heterogeneous bioink compositions. This data-driven approach significantly reduces the experimental workload required to identify bioink compositions conducive to functional tissue growth. It not only streamlines the process of finding the optimal bioink compositions from a vast array of heterogeneous options but also offers a promising avenue for accelerating advancements in tissue engineering by minimizing the need for extensive experimental trials.

Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.

The global demand for an enhanced quality of life and extended lifespan has driven significant advancements in tissue engineering and regenerative medicine. These fields utilize a range of interdisciplinary theories and techniques to repair structurally impaired or damaged tissues and organs, as well as restore their normal functions. Nevertheless, the clinical efficacy of medications, materials, and potent cells used at the laboratory level is always constrained by technological limitations. A novel platform known as adaptable microneedles has been developed to address the abovementioned issues. These microneedles offer a solution for the localized distribution of various cargos while minimizing invasiveness. Microneedles provide favorable patient compliance in clinical settings due to their effective administration and ability to provide a painless and convenient process. In this review article, we summarized the most recent development of microneedles, and we started by classifying various microneedle systems, advantages, and fundamental properties. Subsequently, it provides a comprehensive overview of different types of microneedles, the material used to fabricate microneedles, the fundamental properties of ideal microneedles, and their applications in tissue engineering and regenerative medicine, primarily focusing on preserving and restoring impaired tissues and organs. The limitations and perspectives have been discussed by concluding their future therapeutic applications in tissue engineering and regenerative medicines.