Bioactive composite scaffolds enhance osteoconduction and mineralization, offering potential for bone regeneration. In this study, polysaccharide-based Quince Seed Hydrocolloid (QSH) was combined with Gelatin (Gel), mesoporous bioactive glass nanoparticles (MBGNs), and 45S5 bioactive glass (BG) to fabricate osteoconductive scaffolds. QSH/Gel/BG and QSH/Gel/MBGN composites were characterized for chemical composition, mechanical behavior, and in vitro bioactivity. FTIR and SEM-elemental mapping confirmed homogeneous bioactive glass incorporation. BET analysis revealed a >3-fold increase in surface area for MBGN-containing scaffolds compared to BG and pristine QSH/Gel samples, attributed to the nanoscale mesoporous structure of MBGNs. Swelling tests showed a hydrophilic nature in all scaffolds, with MBGN composites exhibiting the highest swelling ratio (2094 ± 571%), nearly twice that of BG composites (1105 ± 56%). Compression tests indicated similar elastic moduli for MBGN and BG containing scaffolds (2330 and 2140 Pa). Human osteosarcoma cell cultures (28 days) demonstrated high viability (>70%) and osteoconductive response in all composites. Alizarin Red staining and SEM mapping revealed greater mineral accumulation in MBGN-containing scaffolds (Ca/P: 2.53). Overall, both composites supported a 3D osteoconductive microenvironment, while MBGN scaffolds exhibited superior long-term cell viability and mineralization potential, emphasizing their suitability for bone tissue engineering applications.

Three-dimensional (3D) disease modeling and drug screening systems have become important in tissue engineering, drug screening, and development. The newly developed systems support cell and extracellular matrix (ECM) interactions, which are necessary for the formation of the tissue or an accurate model of a disease. Hydrogels are favorable biomaterials due to their properties: biocompatibility, high swelling capacity, tunable viscosity, mechanical properties, and their ability to biomimic the structure and function of ECM. They have been used to model various diseases such as tumors, cancer diseases, neurodegenerative diseases, cardiac diseases, and cardiovascular diseases. Additive manufacturing approaches, such as 3D printing/bioprinting, stereolithography (SLA), selective laser sintering (SLS), and fused deposition modeling (FDM), enable the design of scaffolds with high precision; thus, increasing the accuracy of the disease models. In addition, the aforementioned methodologies improve the design of the hydrogel-based scaffolds, which resemble the complicated structure and intricate microenvironment of tissues or tumors, further advancing the development of therapeutic agents and strategies. Thus, 3D hydrogel-based disease models fabricated through additive manufacturing approaches provide an enhanced 3D microenvironment that empowers personalized medicine toward targeted therapeutics, in accordance with 3D drug screening platforms.

Alzheimer's disease is one of the prevalent neurodegenerative diseases and is characterized by amyloid beta aggregate (Aβ) accumulation. This study reports an Aβ 1-42 induced 3D Alzheimer's disease modeling utilizing differentiated SH-SY5Y spheroids, which is carried out by Magnetic levitation approach, and the neuroprotective effect of Curcumin is further investigated on this model. For this purpose, SH-SY5Y spheroids are differentiated using Retinoic acid-Brain-derived neurotrophic factor sequentially during 3D cell culture. Differentiated spheroids maintained high viability and exhibited significant neuronal characteristics, as evidenced by increasing β-III tubulin and NeuN expressions. 3D Alzheimer's disease model formation and neurotoxicity of Aβ 1-42 aggregates are investigated on un-/differentiated spheroids, resulting in 65% and 51% cell viability, respectively. Characterization of the 3D Alzheimer's disease model is done by immunostaining of Choline acetyltransferase to investigate cholinergic neuron activity loss, showing a 2.2 decrease in fluorescence intensity. Further, Curcumin treatment on the 3D Alzheimer's disease model resulted in augmenting cell viability, confirming neuroprotective effect of Curcumin on Aβ 1-42 induced Alzheimer's disease model. This study highlighted the magnetic levitation-based fabrication of Aβ 1-42-induced 3D Alzheimer's disease model successfully, offering a promising experimental platform for other neurodegenerative disease research and potential clinical applications.

Stem cell imaging and tracking generate a significant impact on regenerative medicine. Among the other techniques, optical imaging methods have emerged as widely used, given their high sensitivity, temporal resolution, and relative accessibility over MRI and PET. Conjugated polymer nanoparticles (CPNs) have emerged as fluorescent probes and have started to stand out due to their high photostability, intense brightness, and flexible design possibilities. They have become promising tools for long-term stem-cell labeling and noninvasive monitoring due to their structural versatility, allowing adjustment of the emission properties and surface functionalities.This chapter provides a comprehensive overview of the role of CPNs in stem cell imaging, their structural and photophysical properties, strategies for cellular labeling, and performance within different optical imaging methods. The representative applications in monitoring migration, proliferation, and differentiation are highlighted, while challenges related to cytotoxicity, biodegradability, and reproducibility are discussed. Emerging approaches, including near-infrared (NIR-II) probes, multimodal systems, and stimuli-responsive designs, are addressed. By summarizing current progress and future directions, this chapter provides a comprehensive outlook on the potential of CPNs to advance next-generation tools for stem cell tracking and regenerative medicine.

Here, a PLLCL-on-chip platform was developed by direct electrospinning of poly (L-lactide-co-ε-caprolactone) (PLLCL) on polymethyl methacrylate (PMMA) microfluidic chips. Designed microchip provides the electrospinning of free-standing aligned PLLCL fibers which eliminates limitations of conventional electrospinning. Besides, aligned fiber structure favors cell alignment through contactless manipulation. Average fiber diameter, and fiber alignment was evaluated by SEM analyses, then, leakage profile of microchip was investigated. 3D cell culture studies were conducted using HeLa and NIH-3T3 cells, and nearly 85

Biomacromolecules derived from natural sources offer superior biocompatibility, biodegradability, and waterholding capacity, which make them promising scaffolds for tissue engineering. Psyllium seed has gained attention in biomedical applications recently due to its gel -forming ability, which is provided by its polysaccharide -rich content consisting mostly of arabinoxylan. This study focuses on the extraction and gelation of Psyllium seed hydrocolloid (PSH) in a single-step water -based protocol, and scaffold fabrication using freezedrying method. After characterization of the scaffold, including morphological, mechanical, swelling, and protein adsorption analyses, 3D cell culture studies were done using NIH-3 T3 fibroblast cells on PSH scaffold, and cell viability was assessed using Live/Dead and Alamar Blue assays. Starting from day 1, high cell viability was obtained, and it reached 90 % at the end of 15 -day culture period. Cellular morphology on PSH scaffold was monitored via SEM analysis; cellular aggregates then spheroid formation were observed throughout the study. Collagen Type -I and F -actin expressions were followed by immunostaining revealing a 9- and 10 -fold increase during long-term culture. Overall, a single-step and non-toxic protocol was developed for extraction and gelation of PSH. Obtained results unveiled that PSH scaffold provided a favorable 3D microenvironment for cells, holding promise for further tissue engineering applications.

Plasmonic sensor platforms are designed for rapid, label-free, and real-time detection and they excel as the next generation biosensors. However, current methods such as Surface Plasmon Resonance require expertise and well-equipped laboratory facilities. Simpler methods such as Localized Surface Plasmon Resonance (LSPR) overcome those limitations, though they lack sensitivity. Hence, sensitivity enhancement plays a crucial role in the future of plasmonic sensor platforms. Herein, a refractive index (RI) sensitivity enhancement methodology is reported utilizing growth of gold nanoparticles (GNPs) on solid support and it is backed up with artificial neural network (ANN) analysis. Sensor platform fabrication was initiated with GNP immobilization onto solid support; immobilized GNPs were then used as seeds for chrono-spectral growth, which was carried out using NH2OH at varied incubation times. The response to RI change of the platform was investigated with varied concentrations of sucrose and ethanol. The detection of bacteria E.coli BL21 was carried out for validation as a model microorganism and results showed that detection was possible at 102 CFU/ml. The data acquired by spectrophotometric measurements were analyzed by ANN and bacteria classification with percentage error rates near 0% was achieved. The proposed LSPR-based, label-free sensor application proved that the developed methodology promises utile sensitivity enhancement potential for similar sensor platforms.

Hydrocolloids, derived from plants, marine, and microbial sources, have become research favorites due to their unique properties. This article provides an overview of the extraction methods, from chemical to enzymatic, to obtain hydrocolloids. Distinctive properties of hydrocolloids, such as high swelling capacity, tunable features, and rapid gelation ability, have gained significant attention recently and have started to be used in tissue engineering and 3D bioprinting. Hydrocolloids will play substantial roles in advancing biomedical products and contributing to improving human health.

The rapid manufacturing of biocomposite scaffold made of saturated-Poly(epsilon-caprolactone) (PCL) and unsaturated Polyester (PE) blends with gelatin and modified gelatin (NCO-Gel) is demonstrated. Polyester blend-based scaffold are fabricated with and without applying potential in the melt electrowriting system. Notably, the applied potential induces phase separation between PCL and PE and drives the formation of PE rich spots at the interface of electrowritten fibers. The objective of the current study is to control the phase separation between saturated and unsaturated polyesters occurring in the melt electro-writing process and utilization of this phenomenon to improve efficiency of biofunctionalization at the interface of scaffold via Aza-Michael addition reaction. Electron-deficient triple bonds of PE spots on the fibers exhibit good potential for the biofunctionalization via the aza-Michael addition reaction. PE spots are found to be pronounced in which blend compositions are PCLPE as 90:10 and 75:25 %. The biofunctionalization of scaffold is monitored through C-N bond formation appeared at 400 eV via X-ray photoelectron spectroscopy (XPS) and XPS chemical mapping. The described biofunctionalization methodology suggest avoiding use of multi-step chemical modification on additive manufacturing products and thereby rapid prototyping of functional polymer blend based scaffolds with enhanced biocompatibility and preserved mechanical properties. Additionally one-step additive manufacturing method eliminates side effects of toxic solvents and long modification steps during scaffold fabrication.

Magnetic levitation (MagLev) is a powerful and versatile technique that can sort objects based on their density differences. This paper reports the sorting of SH-SY5Y cells for neuronal differentiation by the MagLev technique. Herein, SH-SY5Y cells were differentiated with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF). Neuronal differentiation was confirmed by neurite extension measurement and the immunostaining assay. Neurites reached the maximum length on day 9 after sequential treatment with RA-BDNF. Neuronal marker expression of un-/differentiated cells was investigated by β-III tubulin and neuronal nuclei (NeuN) and differentiated cells exhibited a higher fluorescence intensity compared to un-/differentiated cells. MagLev results revealed that the density of differentiated SH-SY5Y cells gradually increased from 1.04 to 1.06 g/mL, while it remained stable at 1.05 g/mL for un-/differentiated cells. These findings signified that cell density would be a potent indicator of neuronal differentiation. Overall, it was shown that MagLev methodology can provide rapid, label-free, and easy sorting to analyze the differentiation of cells at a single-cell level.

Alginate forms a hydrogel via physical cross-linking with divalent cations. In literature, Ca2+ is mostly utilized due to strong interactions but additional procedures are required to disassociate Ca-alginate hydrogels. On the other hand, Mg-alginate hydrogels disassociate spontaneously, which might benefit certain applications. This study introduces Mg-alginate as the main component of a bio-ink for the first time to obtain 3D tumor models by magnetic bio-patterning technique. The bio-ink contains magnetic nanoparticles (MNPs) for magnetic manipulation, Mg-alginate hydrogel as a sacrificial material, and cells. The applicability of the methodology is tested for the formation of 3D tumor models using HeLa, SaOS-2, and SH-SY5Y cells. Long-term cultures are examined by Live/dead and MTT analysis and revealed high cell viability. Subsequently, Collagen and F-actin expressions are observed successfully in 3D tumor models. Finally, the anti-cancer drug Doxorubicin (DOX) effect is investigated on 3D tumor models, and IC50 values is calculated to assess the drug response. As a result, significantly higher drug resistance is observed for bio-patterned 3D tumor models up to tenfold compared to 2D control. Overall, Mg-alginate hydrogel is successfully used to form bio-patterned 3D tumor models, and the applicability of the model is shown effectively, especially as a drug screening platform.

This study describes the formation, size control, and penetration behavior of polymer nanodots (Pdots) consisting of single or few chain polythiophene-based conjugated polyelectrolytes (CPEs) via nanophase separation between good solvent and poor solvent of CPE. Though the chain singularity may be associated with dilution nanophase separation suggests that molecules of a good solvent create a thermodynamically driven solvation layer surrounding the CPEs and thereby separating the single chains even in their poor solvents. This statement is therefore corroborated with emission intensity/lifetime, particle size, and scattering intensity of polyelectrolyte in good and poor solvents. Regarding the augmented features, Pdots are implemented into cell imaging studies to understand the nuclear penetration and to differentiate the invasive characteristics of breast cancer cells. The python based red, green, blue (RGB) color analysis   depicts that Pdots have more nuclear penetration ability in triple negative breast cancer cells due to the different nuclear morphology in shape and composition and Pdots have penetrated cell membrane as well as extracellular matrix in spheroid models. The current Pdot protocol and its utilization in cancer cell imaging are holding great promise for gene/drug delivery to target cancer cells by explicitly achieving the very first priority of nuclear intake.

Pathogenic microorganisms impose great risk especially in resource-limited settings due to inaccessibility of diagnostic tools and monitoring devices. This is mainly caused by current methods often being economically demanding and complex in practice; while these methods are sensitive and accurate, they rarely follow Point-of-care (POC) approaches, which is essential for rapid detection and intervention. Incorporating origami into paper-based analytical devices (μPAD) presents an innovative alternative, offering affordability, portability, and ease of disposal. Herein, a colorimetric origami μPAD that is suitable for use in POC applications was developed. The μPAD was fabricated via laser ablation utilizing PVDF and cellulose membranes. In order to develop the biosensor platform, fabrication parameters were optimized and hydrophilicity of PVDF membranes was improved using various solvents. The PVDF membranes were characterized through light microscopy imaging, protein adsorption assay and contact angle measurements. Then, optimization of the assay parameters was carried out in order to improve sensitivity and resolution of the μPAD, utilizing Box-Behnken experimental design. The responses generated by the origami μPAD in form of visible color development were then analyzed using image processing. After optimization is concluded, E. coli detection was carried out as a model system. Resulting calculations showed a limit of detection (LoD) of 2 CFU/mL and a dynamic working range up to 106 CFU/mL for E. coli. Overall, developed origami μPAD promises an economic advantage compared to conventional methods, and provides rapid and sensitive results without the requirement of expertise or complex equipment.

Extracellular vesicles, especially exosomes, have attracted attention in the last few decades as novel cancer biomarkers. Exosomal membrane proteins provide easy-to-reach targets and can be utilized as information sources of their parent cells. In this study, a MagLev-based, highly sensitive, and versatile biosensor platform for detecting minor differences in the density of suspended objects is proposed for exosome detection. The developed platform utilizes antibody-functionalized microspheres to capture exosomal membrane proteins (ExoMPs) EpCAM, CD81, and CD151 as markers for cancerous exosomes, exosomes, and non-small cell lung cancer (NSCLC)-derived exosomes, respectively. Initially, the platform was utilized for protein detection and quantification by targeting solubilized ExoMPs, and a dynamic range of 1-100 nM, with LoD values of 1.324, 0.638, and 0.722 nM for EpCAM, CD81, and CD151, were observed, respectively. Then, the sensor platform was tested using exosome isolates derived from NSCLC cell line A549 and MRC5 healthy lung fibroblast cell line. It was shown that the sensor platform is able to detect and differentiate exosomal biomarkers derived from cancerous and non-cancerous cell lines. Overall, this innovative, simple, and rapid method shows great potential for the early diagnosis of lung cancer through exosomal biomarker detection.

AbstractThis study describes the formation of single‐chain polymer dots (Pdots) via ultrasonic emulsification of nonionic donor‐acceptor‐donor type (D–A–D) alkoxy thiophene–benzobisthiadiazole‐based conjugated polymers (Poly BT) with amphiphilic cetyltrimethylammonium bromide (CTAB). The methodology yields Pdots with a high cationic surface charge (+56.5 mV ± 9.5) and average hydrodynamic radius of 12 nm. Optical characterization reveals that these Pdots emit near‐infrared (NIR) light at a maximum wavelength of 860 nm owing to their conjugated polymer backbone consisting of D–A–D monomers. Both colloidal and optical properties of these Pdots make them promising fluorescence emissive probes for bioimaging applications. The significant advantage of positively charged Pdots is demonstrated in diffusion‐limited mediums such as tissues, utilizing human epithelial breast adenocarcinoma, ATCC HTB‐22 (MCF‐7), human bone marrow neuroblastoma, ATCC CRL‐2266 (SH‐SY5Y), and rat adrenal gland pheochromocytoma, CRL‐1721 (PC‐12) tumor spheroid models. Fluorescence microscopy analysis of tumor spheroids from MCF‐7, SH‐SY5Y, and PC‐12 cell lines reveals the intensity profile of Pdots, confirming extensive penetration into the central regions of the models. Moreover, a comparison with mitochondria staining dye reveals an overlap between the regions stained by Pdots and the dye in all three tumor spheroid models. These results suggest that single‐chain D–A–D type Pdots, cationized via CTAB, exhibit long‐range mean free path of penetration (≈1 µm) in dense mediums and tumors.

Plant-derived hydrocolloids offer promising prospects in biomedical applications. Among these, Flaxseed hydrocolloid (FSH) can form a soft, elastic, and biocompatible hydrocolloid with tunable viscosity and superior swelling capacity, making it an attractive scaffold. This study introduces a green extraction method for FSH, employing a single-step aqueous extraction process and fabrication of FSH scaffold. Despite growing interest, the pristine form of FSH has not been investigated for sustainable long-term three-dimensional (3D) cell culture. Here, FSH scaffolds were thoroughly characterized for their morphological, chemical, mechanical, and biological properties. 3D cell culture experiments were conducted using NIH-3T3 mouse fibroblast cells, and cell viability was assessed using live/dead and Alamar Blue assays. High cell viability was sustained for long term compared with 2D cell culture. Cell adhesion and 3D cellular morphology on FSH scaffold for 30 days were monitored by scanning electron microscopy analysis. Also, collagen type-I and F-actin expressions were analyzed by immunostaining after 30 days of culture, resulting in 5- and 4-fold increments of fluorescence intensity, respectively. Results indicate sustained cell viability in the long term and favorable cell-material interaction, demonstrating the potential of FSH as a scaffold. This study emphasizes the importance of the green extraction approach, improving the biocompatibility and functionality of FSH tissue engineering applications.

Paper-based microfluidics is an emerging analysis tool used in various applications, especially in point-of-care (PoC) diagnostic applications, due to its advantages over other types of microfluidic devices in terms of simplicity in both production and operation, cost-effectiveness, rapid response time, low sample consumption, biocompatibility, and ease of disposal. Recently, various techniques have been developed and utilized for the fabrication of paper-based microfluidics, such as photolithography, micro-embossing, wax and PDMS printing, etc. In this study, we offer a fabrication methodology for a microfluidic paper-based immunosorbent assay (μPISA) platform and the detection of Hepatitis C Virus (HCV) was carried out to validate this platform. A laser ablation technique was utilized to form hydrophobic barriers easily and rapidly, which was the major advantage of the developed fabrication methodology. The characterization of the μPISA platform was performed in terms of micro-channel properties using bright-field (BF) microscopy, and surface properties using scanning electron microscopy (SEM). At the same time, sample volume and liquid handling capacity were analyzed quantitatively. Ablation speed (S) and laser power (P) were optimized, and it was shown that one combination (10P60S) provided minimal deviation in micro-channel dimensions and prevented deterioration of hydrophobic barriers. Also, the minimum hydrophobic barrier width, which prevents cross-barrier bleeding, was determined to be 255.92 ± 10.01 μm. Furthermore, colorimetric HCV NS3 detection was implemented to optimize and validate the μPISA platform. Here, HCV NS3 in both PBS and human blood plasma was successfully detected by the naked eye at concentrations as low as 1 ng mL-1 and 10 ng mL-1, respectively. Moreover, the limit of detection (LoD) values for HCV NS3 were acquired as 0.796 ng mL-1 in PBS and 2.203 ng mL-1 in human blood plasma with a turnaround time of 90 min. In comparison with conventional ELISA, highly sensitive and rapid HCV NS3 detection was accomplished colorimetrically on the developed μPISA platform.

Impact statement Contactless manipulation and cell patterning techniques provide rapid and cost-effective three-dimensional (3D) cell culture model formation for tissue engineering applications. The present study introduces a new methodology that comprised alginate-based bioink to pattern cells via contactless magnetic manipulation to fabricate 3D cardiac structures. The developed cardiac model was evaluated in terms of Doxorubicin-induced cardiotoxicity and biopatterned 3D cardiac structures were found more resistant to drug exposure compared to two-dimensional control.

Three-dimensional (3D) cell culture offers alternative approaches to two-dimensional (2D) cell culture and animal models because it mimics the physiology and metabolism of native tissues. Magnetic levitation (MagLev) is a new and promising technique for biofabrication of 3D cardiac spheroids in a scaffold-free microenvironment. In this study, we introduce a rapid and cost-effective methodology for the fabrication of 3D cardiac spheroids to investigate doxorubicin-induced cardiotoxicity. Here, Gadobutrol (Gx) was utilized as a paramagnetic agent to magnetically assemble cardiac cells. For this purpose, initial cell density, and culture time were optimized. Then, fabricated 3D cardiac spheroids were characterized by immunostaining of cellular and extracellular components. In addition, cardiac-specific markers of cardiac troponin T and MYH6 were investigated. Further, the drug response of 3D cardiac spheroids was evaluated for doxorubicin, and IC values were obtained as 3.5 and 14.7 µM for 2D control and 3D cardiac spheroids, respectively. The acquired results emphasize that 3D cardiac spheroids are more resistant to drug exposure. Hereby, these findings indicate that biofabrication of 3D cardiac spheroids via MagLev methodology might be an alternative platform for drug screening.