Programming temporal stiffness cues within extracellular matrix hydrogels for modelling cancer niches.

Extracellular matrix (ECM) stiffening is a common occurrence during the progression of many diseases, such as breast cancer. To accurately mimic the pathophysiological context of disease within 3D in vitro models, there is high demand for smart biomaterials which replicate the dynamic and temporal mechanical cues of diseased states. This study describes a preclinical disease model, using breast cancer as an example, which replicates the dynamic plasticity of the tumour microenvironment by incorporating temporal (3-week progression) biomechanical cues within a tissue-specific hydrogel microenvironment. The composite hydrogel formulation, integrating adipose-derived decellularised ECM (AdECM) and silk fibroin, was initially crosslinked using a visible light-mediated system, and then progressively stiffened through spontaneous secondary structure interactions inherent between the polymer chains (∼10-15 kPa increase, with a final stiffness of 25 kPa). When encapsulated and cultured in vitro, MCF-7 breast cancer cells initially formed numerous, large spheroids (>1000 µm2 in area), however, with progressive temporal stiffening, cells demonstrated growth arrest and underwent phenotypic changes resulting in intratumoral heterogeneity. Unlike widely-investigated static mechanical models, this stiffening hydrogel allowed for progressive phenotypic changes to be observed, and fostered the development of mature organoid-like spheroids, which mimicked both the organisation and acinar-structures of mature breast epithelium. The spheroids contained a central population of cells which expressed aggressive cellular programs, evidenced by increased fibronectin expression and reduction of E-cadherin. The phenotypic heterogeneity observed using this model is more reflective of physiological tumours, demonstrating the importance of establishing temporal cues within preclinical models in future work. Overall, the developed model demonstrated a novel strategy to uncouple ECM biomechanical properties from the cellular complexities of the disease microenvironment and offers the potential for wide applicability in other 3D in vitro disease models through addition of tissue-specific dECM materials.

Engineering of Uniform Epidermal Layers via Sacrificial Gelatin Bioink-Assisted 3D Extrusion Bioprinting of Skin.

To reconstruct an ideal full-thickness skin model, basal keratinocytes must be distributed as a confluent monolayer on the dermis. However, the currently available extrusion bioprinting method for the skin is limited when producing an air-exposed cellular monolayer because the cells are encapsulated within a bioink. This is the first study to use sacrificial gelatin-assisted extrusion bioprinting to reproduce a uniform and stratified epidermal layer. Experimental analyses of the rheological properties, printability, cell viability, and initial keratinocyte adhesion shows that the optimal gelatin bioink concentration is 4 wt.%. The appropriate thickness of the bioprinted gelatin structure for achieving a confluent keratinocyte layer is determined to be 400 µm. The suggested strategy generates a uniform keratinocyte monolayer with tight junctions throughout the central and peripheral regions, whereas manual seeding generates non-uniform cellular aggregates and vacancies. These results influence gene expression, exhibiting a propensity for epidermal differentiation. Finally, the gelatin-assisted keratinocytes are bioprinted onto a dermis composed of gelatin methacryloyl and dermis-derived decellularized extracellular matrix to establish a full-thickness skin model. Thus, this strategy leads to significant improvements in epidermal differentiation/stratification. The findings demonstrate that the gelatin-assisted approach is advantageous for recreating reliable full-thickness skin models with significant consistency for mass production.

3D biofabrication of diseased human skin models in vitro.

Human skin is an organ located in the outermost part of the body; thus, it frequently exhibits visible signs of physiological health. Ethical concerns and genetic differences in conventional animal studies have increased the need for alternative in vitro platforms that mimic the structural and functional hallmarks of natural skin. Despite significant advances in in vitro skin modeling over the past few decades, different reproducible biofabrication strategies are required to reproduce the pathological features of diseased human skin compared to those used for healthy-skin models. To explain human skin modeling with pathological hallmarks, we first summarize the structural and functional characteristics of healthy human skin. We then provide an extensive overview of how to recreate diseased human skin models in vitro, including models for wounded, diabetic, skin-cancer, atopic, and other pathological skin types. We conclude with an outlook on diseased-skin modeling and its technical perspective for the further development of skin engineering.

Blood-Lymphatic Integrated System with Heterogeneous Melanoma Spheroids via In-Bath Three-Dimensional Bioprinting for Modelling of Combinational Targeted Therapy.

Although metastatic melanoma can be managed with chemotherapy, its heterogeneity and resistance to therapy remain poorly understood. In addition to the spread of melanoma in the bloodstream, melanoma-stroma interaction and the lymphatic system play active roles in said heterogeneity and resistance, leading to its progression and metastasis. Reproducing the complexities of the melanoma microenvironment in vitro will help understanding its progression and enhance the translatability of potential cancer therapeutics. A blood-lymphatic integrated system with heterogeneous melanoma spheroids (BLISH) using the in-bath bioprinting process is developed. The process uniformly prints size-controllable metastatic melanoma spheroids along with biomimetic blood and lymphatic vessels (LVs). The system reproduces hallmark events of metastatic melanoma, such as tumor stroma interaction, melanoma invasion, and intravasation. The application of the system to investigate the anticancer effect of combinational targeted therapy suggests that it can be used to study the pathophysiology of melanoma and improve the accuracy of drug response monitoring in skin cancer.

Electrochemical Impedance-Based Biosensors for the Label-Free Detection of the Nucleocapsid Protein from SARS-CoV-2.

Diagnosis of coronavirus disease (COVID-19) is important because of the emergence and global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Real-time polymerase chain reaction (PCR) is widely used to diagnose COVID-19, but it is time-consuming and requires sending samples to test centers. Thus, the need to detect antigens for rapid on-site diagnosis rather than PCR is increasing. We quantified the nucleocapsid (N) protein in SARS-CoV-2 using an electro-immunosorbent assay (El-ISA) and a multichannel impedance analyzer with a 96-interdigitated microelectrode sensor (ToAD). The El-ISA measures impedance signals from residual detection antibodies after sandwich assays and thus offers highly specific, label-free detection of the N protein with low cross-reactivity. The ToAD sensor enables the real-time electrochemical detection of multiple samples in conventional 96-well plates. The limit of detection for the N protein was 0.1 ng/mL with a detection range up to 10 ng/mL. This system did not detect signals for the S protein. While this study focused on detecting the N protein in SARS-CoV-2, our system can also be widely applicable to detecting various biomolecules involved in antigen-antibody interactions.

Construction of Tissue-Level Cancer-Vascular Model with High-Precision Position Control via In Situ 3D Cell Printing.

During tumor progression, the size and location of the tumor are important factors closely associated with the metastatic potential of the cancer as they largely govern tumor hypoxia and angiogenesis. However, despite the achievements of previous studies, these critical factors are poorly studied, mainly due to the lack of a flexible technique that can readily control 3D tumor mimicking constructs and their spatial relations with vasculature. Here, a novel tissue-level platform consisting of a metastatic cancer unit (MCU) and a perfusable vascular endothelium system (VES) is presented using in situ 3D cell printing. Size-tunable and position-controllable 3D cancer spheroids (500-1000 µm) are directly printed within the established bath bioink with a self-driven perfusable vascular channel. The cancer-vascular interactions are generated through controlling the distance between MCU and VES to investigate metastasis-associated changes at adjacent and distal regions. The result shows that MCU in 600 µm diameter includes hypoxia, invasion, and angiogenetic signaling. The further observations demonstrate that the proximity of MCU to VES augments the epithelial-mesenchymal transition (EMT) in MCU and vascular dysfunction/inflammation in VES, corroborating the positional significance in tumor metastasis. The platform with the precise-positioning control enables the recapitulation of patient's detailed metastatic progression, opening the chance for precision cancer medicine.

3D Printing of Pharmaceutical Application: Drug Screening and Drug Delivery.

Advances in three-dimensional (3D) printing techniques and the development of tailored biomaterials have facilitated the precise fabrication of biological components and complex 3D geometrics over the past few decades. Moreover, the notable growth of 3D printing has facilitated pharmaceutical applications, enabling the development of customized drug screening and drug delivery systems for individual patients, breaking away from conventional approaches that primarily rely on transgenic animal experiments and mass production. This review provides an extensive overview of 3D printing research applied to drug screening and drug delivery systems that represent pharmaceutical applications. We classify several elements required by each application for advanced pharmaceutical techniques and briefly describe state-of-the-art 3D printing technology consisting of cells, bioinks, and printing strategies that satisfy requirements. Furthermore, we discuss the limitations of traditional approaches by providing concrete examples of drug screening (organoid, organ-on-a-chip, and tissue/organ equivalent) and drug delivery systems (oral/vaginal/rectal and transdermal/surgical drug delivery), followed by the introduction of recent pharmaceutical investigations using 3D printing-based strategies to overcome these challenges.

Improved chondrogenic performance with protective tracheal design of Chitosan membrane surrounding 3D-printed trachea.

In recent tracheal tissue engineering, limitations in cartilage reconstruction, caused by immature delivery of chondrocyte-laden components, have been reported beyond the complete epithelialization and integration of the tracheal substitutes with the host tissue. In an attempt to overcome such limitations, this article introduces a protective design of tissue-engineered trachea (TraCHIM) composed of a chitosan-based nanofiber membrane (CHIM) and a 3D-printed biotracheal construct. The CHIM was created from chitosan and polycaprolactone (PCL) using an electrospinning process. Upon addition of chitosan to PCL, the diameter of electrospun fibers became thinner, allowing them to be stacked more closely, thereby improving its mechanical properties. Chitosan also enhances the hydrophilicity of the membranes, preventing them from slipping and delaminating over the cell-laden bioink of the biotracheal graft, as well as protecting the construct. Two weeks after implantation in Sprague-Dawley male rats, the group with the TraCHIM exhibited a higher number of chondrocytes, with enhanced chondrogenic performance, than the control group without the membrane. This study successfully demonstrates enhanced chondrogenic performance of TraCHIM in vivo. The protective design of TraCHIM opens a new avenue in engineered tissue research, which requires faster tissue formation from 3D biodegradable materials, to achieve complete replacement of diseased tissue.

Engineering of diseased human skin equivalent using 3D cell printing for representing pathophysiological hallmarks of type 2 diabetes in vitro.

Despite many significant advances in 3D cell printing for skin, a disease model displaying the pathological processes present in the native skin has not been reported yet. Therefore, we were motivated for modeling a 3D diseased skin tissue with pathophysiological hallmarks of type 2 diabetes in vitro based on 3D cell printing technique. By stimulating epidermal-dermal intercellular crosstalk found in the native skin, it was hypothesized that normal keratinocytes would be differentiated as diabetic epidermis when interacting with the diabetic dermal compartment. To prove this, a novel wounded skin model was successfully devised during tissue maturation in vitro. Interestingly, the slow re-epithelization was observed in our diabetic model, which is a representative hallmark of diabetic skin. Using the versatility of 3D cell printing, the structural similarities and diabetic properties of the model were further augmented by addition of perfusable vascularized diabetic hypodermis. Insulin resistance, adipocyte hypertrophy, inflammatory reactions, and vascular dysfunction, as the typical hallmarks in diabetes, were found under hyperglycemia. Finally, the feasibility of this new disease model for drug development was successfully demonstrated through application of test drugs. We trust that this study provides a pioneering step towards 3D cell printing-based in vitro skin disease modeling.

Nanomaterial-based Optical and Electrochemical Biosensors for Amyloid beta and Tau: Potential for early diagnosis of Alzheimer's Disease.

INTRODUCTION: Alzheimer's disease (AD), a heterogeneous pathological process representing the most common causes of dementia worldwide, has required early and accurate diagnostic tools. Neuropathological hallmarks of AD involve the aberrant accumulation of Amyloid beta (Aß) into Amyloid plaques and hyperphosphorylated Tau into neurofibrillary tangles, occurring long before the onset of brain dysfunction.Areas covered:Considering the significance of Aß and Tau in AD pathogenesis, these proteins have been adopted as core biomarkers of AD, and their quantification has provided precise diagnostic information to develop next-generation AD therapeutic approaches. However, conventional diagnostic methods may not suffice to achieve clinical criteria that are acceptable for proper diagnosis and treatment. The advantages of nanomaterial-based biosensors including facile miniaturization, mass fabrication, ultra-sensitivity, make them useful to be promising tools to measure Aß and Tau simultaneously for accurate validation of low-abundance yet potentially informative biomarkers of AD.. EXPERT OPINION: The study has identified the potential application of advanced biosensors as standardized clinical diagnostic tools for AD, evolving the way for new and efficient AD control with minimum economic and social burden. After clinical trial, nanobiosensors for measuring Aß and Tau simultaneously possess innovative diagnosis of AD to provide significant contributions to primary Alzheimer's care intervention.

Flexible Adipose-Vascular Tissue Assembly Using Combinational 3D Printing for Volume-Stable Soft Tissue Reconstruction.

A new concept, assembling cell-laden tissue modules, is for the first time proposed for soft tissue engineering. Adipose-vascular tissue modules composed of a synthetic polymer-based substructure and customized bioinks using planar 3D cell printing are engineered. Such tissue modules are systematically assembled into a synthetic polymer-based module holder fabricated with rotational 3D printing, resulting in the development of a flexible and volumetric tissue assembly. Whereas most of the previous studies about the construction of adipose tissue are limited to hypoxia, poor vascularization, rapid resorption, and mismatch in mechanical properties, it is aimed to realize the construction of nonhypoxic, flexible, and volume-stable tissue assembly in this study. The significance of engineered tissue assembly is proven through various in vitro and in vivo evaluations. In particular, stable volume and remarkable neovascularization/adipogenesis are observed in the implanted assembly over four weeks. Interestingly, the size of newly formed lipid droplets and the remodeled morphology in the assembly are comparable to those in native adipose tissue. As far as it is known, this work is a first report suggesting a cell printing-based tissue assembly for functional reconstruction of soft tissue.

Nanobiosensors for Non-Amyloidbeta-Tau Biomarkers as Advanced Reporters of Alzheimer's Disease.

Emerging nanomaterials providing benefits in sensitivity, specificity and cost-effectiveness are being widely investigated for biosensors in the application of Alzheimer's disease (AD) diagnosis. Core biomarkers amyloid-beta (Aß) and Tau have been considered as key neuropathological hallmarks of AD. However, they did not sufficiently reflect clinical severity and therapeutic response, proving the difficulty of the Aß- and Tau-targeting therapies in clinical trials. In recent years, there has still been a shortage of sensors for non-Aß-Tau pathophysiological biomarkers that serve as advanced reporters for the early diagnosis of AD, predict AD progression, and monitor the treatment response. Nanomaterial-based sensors measuring multiple non-Aß-Tau biomarkers could improve the capacity of AD progression characterization and supervised treatment, facilitating the comprehensive management of AD. This is the first review to principally represent current nanobiosensors for non-Aß-Tau biomarker and that strategically deliberates future perspectives on the merit of non-Aß-Tau biomarkers, in combination with Aß and Tau, for the accurate diagnosis and prognosis of AD.

Comparative analyses of plasma amyloid-ß levels in heterogeneous and monomerized states by interdigitated microelectrode sensor system.

Detection of amyloid-ß (Aß) aggregates contributes to the diagnosis of Alzheimer disease (AD). Plasma Aß is deemed a less invasive and more accessible hallmark of AD, as Aß can penetrate blood-brain barriers. However, correlations between biofluidic Aß concentrations and AD progression has been tenuous. Here, we introduce a diagnostic technique that compares the heterogeneous and the monomerized states of Aß in plasma. We used a small molecule, EPPS [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid], to dissociate aggregated Aß into monomers to enhance quantification accuracy. Subsequently, Aß levels of EPPS-treated plasma were compared to those of untreated samples to minimize inter- and intraindividual variations. The interdigitated microelectrode sensor system was used to measure plasma Aß levels on a scale of 0.1 pg/ml. The implementation of this self-standard blood test resulted in substantial distinctions between patients with AD and individuals with normal cognition (NC), with selectivity and sensitivity over 90%.

Electrochemical Detection of C-Reactive Protein in Human Serum Based on Self-Assembled Monolayer-Modified Interdigitated Wave-Shaped Electrode.

An electrochemical capacitance immunosensor based on an interdigitated wave-shaped micro electrode array (IDWµE) for direct and label-free detection of C-reactive protein (CRP) was reported. A self-assembled monolayer (SAM) of dithiobis (succinimidyl propionate) (DTSP) was used to modify the electrode array for antibody immobilization. The SAM functionalized electrode array was characterized morphologically by atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX). The nature of gold-sulfur interactions on SAM-treated electrode array was probed by X-ray photoelectron spectroscopy (XPS). The covalent linking of anti-CRP-antibodies onto the SAM modified electrode array was characterized morphologically through AFM, and electrochemically through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The application of phosphate-buffered saline (PBS) and human serum (HS) samples containing different concentrations of CRP in the electrode array caused changes in the electrode interfacial capacitance upon CRP binding. CRP concentrations in PBS and HS were determined quantitatively by measuring the change in capacitance (ΔC) through EIS. The electrode immobilized with anti-CRP-antibodies showed an increase in ΔC with the addition of CRP concentrations over a range of 0.01-10,000 ng mL-1. The electrode showed detection limits of 0.025 ng mL-1 and 0.23 ng mL-1 (S/N = 3) in PBS and HS, respectively. The biosensor showed a good reproducibility (relative standard deviation (RSD), 1.70%), repeatability (RSD, 1.95%), and adequate selectivity in presence of interferents towards CRP detection. The sensor also exhibited a significant storage stability of 2 weeks at 4 °C in 1× PBS.

A highly sensitive plasma-based amyloid-ß detection system through medium-changing and noise cancellation system for early diagnosis of the Alzheimer's disease.

We developed an interdigitated microelectrode (IME) sensor system for blood-based Alzheimer's disease (AD) diagnosis based on impedimetric detection of amyloid-ß (Aß) protein, which is a representative candidate biomarker for AD. The IME sensing device was fabricated using a surface micromachining process. For highly sensitive detection of several tens to hundreds of picogram/mL of Aß in blood, medium change from plasma to PBS buffer was utilized with signal cancellation and amplification processing (SCAP) system. The system demonstrated approximately 100-folds higher sensitivity according to the concentrations. A robust antibody-immobilization process was used for stability during medium change. Selectivity of the reaction due to the affinity of Aß to the antibody and the sensitivity according to the concentration of Aß were also demonstrated. Considering these basic characteristics of the IME sensor system, the medium change was optimized in relation to the absolute value of impedance change and differentiated impedance changes for real plasma based Aß detection. Finally, the detection of Aß levels in transgenic and wild-type mouse plasma samples was accomplished with the designed sensor system and the medium-changing method. The results confirmed the potential of this system to discriminate between patients and healthy controls, which would enable blood-based AD diagnosis.