Advances in microfabrication technologies in tissue engineering and regenerative medicine.

BACKGROUND: Tissue engineering provides various strategies to fabricate an appropriate microenvironment to support the repair and regeneration of lost or damaged tissues. In this matter, several technologies have been implemented to construct close-to-native three-dimensional structures at numerous physiological scales, which are essential to confer the functional characteristics of living tissues. METHODS: In this article, we review a variety of microfabrication technologies that are currently utilized for several tissue engineering applications, such as soft lithography, microneedles, templated and self-assembly of microstructures, microfluidics, fiber spinning, and bioprinting. RESULTS: These technologies have considerably helped us to precisely manipulate cells or cellular constructs for the fabrication of biomimetic tissues and organs. Although currently available tissues still lack some crucial functionalities, including vascular networks, innervation, and lymphatic system, microfabrication strategies are being proposed to overcome these issues. Moreover, the microfabrication techniques that have progressed to the preclinical stage are also discussed. CONCLUSIONS: This article aims to highlight the advantages and drawbacks of each technique and areas of further research for a more comprehensive and evolving understanding of microfabrication techniques in terms of tissue engineering and regenerative medicine applications.

Micro and Nanoscale Technologies for Diagnosis of Viral Infections.

Viral infection is one of the leading causes of mortality worldwide. The growth of globalization significantly increases the risk of virus spreading, making it a global threat to future public health. In particular, the ongoing coronavirus disease 2019 (COVID-19) pandemic outbreak emphasizes the importance of devices and methods for rapid, sensitive, and cost-effective diagnosis of viral infections in the early stages by which their quick and global spread can be controlled. Micro and nanoscale technologies have attracted tremendous attention in recent years for a variety of medical and biological applications, especially in developing diagnostic platforms for rapid and accurate detection of viral diseases. This review addresses advances of microneedles, microchip-based integrated platforms, and nano- and microparticles for sampling, sample processing, enrichment, amplification, and detection of viral particles and antigens related to the diagnosis of viral diseases. Additionally, methods for the fabrication of microchip-based devices and commercially used devices are described. Finally, challenges and prospects on the development of micro and nanotechnologies for the early diagnosis of viral diseases are highlighted.

Development of QCM based biosensor for the selective and sensitive detection of paraoxon.

In this study, a quartz crystal microbalance (QCM) based sensor was developed that selectively recognizes and binds the paraoxon molecule. For this purpose, poly (styrene-maleic anhydride) (PSMA) polymer was synthesized to obtain nanofiber. A 30% (wt/wt) PSMA/DMSO solution was prepared for use in the electrospinning process, with operating conditions of 5 kV potential and 1 mL/h flow rate. After obtaining the nanofibers, AChE enzyme was immobilized to nanofiber. The QCM gold electrode surface was coated with AChE immobilized nanofiber. For this purpose, the QCM electrode was first functionalized with 4-aminothiophenol. The quartz electrode coated with the recognition layer was sequentially studied with aqueous solutions containing 50% (v/v) methanol, ranging from 0.1 ppm to 10 ppm of paraoxon. When the electrode interacts with paraoxon, the frequency value decreases. The obtained data were converted to graphs and a calibration graph was obtained. The LOD value was found to be 4.57 × 10-8 and the LOQ value was found to be 1.52 × 10-7 M. These results show us that the developed method can analyze very small quantities of paraoxon samples. The Langmuir adsorption equation was used to study the interaction of the bond between the electrode surface and the paraoxon. For the calculation of the coupling constant Ka, Δm/C (g/M) versus Δm (g) was plotted on the graph. The Ka binding constant of the obtained graphic was found to be 5 × 107 M-1.

New synthesis method for 4-MAPBA monomer and using for the recognition of IgM and mannose with MIP-based QCM sensors.

Quartz crystal microbalance (QCM) sensors coated with molecularly imprinted polymers (MIP) have been developed for the recognition of immunoglobulin M (IgM) and mannose. In this method, methacryloylamidophenylboronic acid (MAPBA) was used as a monomer and mannose was used as a template. For this purpose, initially, QCM electrodes were modified with 2-propene-1-thiol to form mannose-binding regions on the QCM sensor surface. In the second step, the methacryloylamidophenylboronic acid-mannose [MAPBA-mannose], pre-organized monomer system, was prepared using the MAPBA monomer. Then, a molecularly imprinted film was coated on to the QCM electrode surface under UV light using ethylene glycol dimethacrylate (EDMA), and azobisisobutyronitrile (AIBN) as a cross-linking agent and an initiator, respectively. The mannose can be simultaneously bound to MAPBA and fitted into the shape-selective cavities. The binding affinity of the mannose-imprinted sensors was investigated using the Langmuir isotherm. The mannose-imprinted QCM electrodes have shown homogeneous binding sites for mannose (K(a): 3.3 × 10(4) M(-1)) and heterogeneous binding sites for IgM (K(a1): 1.0 × 10(4) M(-1); K(a2): 3.3 × 10(3) M(-1)).

Biogenic Synthesis of Multifunctional Silver Oxide Nanoparticles (Ag2ONPs) Using Parieteria alsinaefolia Delile Aqueous Extract and Assessment of Their Diverse Biological Applications.

Green nanotechnology has made the synthesis of nanoparticles a possible approach. Nanotechnology has a significant impact on several scientific domains and has diverse applications in different commercial areas. The current study aimed to develop a novel and green approach for the biosynthesis of silver oxide nanoparticles (Ag2ONPs) utilizing Parieteria alsinaefolia leaves extract as a reducing, stabilizing and capping agent. The change in color of the reaction mixture from light brown to reddish black determines the synthesis of Ag2ONPs. Further, different techniques were used to confirm the synthesis of Ag2ONPs, including UV-Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), Energy-dispersive X-ray spectroscopy (EDX), zeta potential and dynamic light scattering (DLS) analyses. The Scherrer equation determined a mean crystallite size of ~22.23 nm for Ag2ONPs. Additionally, different in vitro biological activities have been investigated and determined significant therapeutic potentials. Radical scavenging DPPH assay (79.4%), reducing power assay (62.68 ± 1.77%) and total antioxidant capacity (87.5 ± 4.8%) were evaluated to assess the antioxidative potential of Ag2ONPs. The disc diffusion method was adopted to evaluate the antibacterial and antifungal potentials of Ag2ONPs using different concentrations (125-1000 µg/mL). Moreover, the brine shrimp cytotoxicity assay was investigated and the LC50 value was calculated as 2.21 µg/mL. The biocompatibility assay using red blood cells (<200 µg/mL) confirmed the biosafe and biocompatible nature of Ag2ONPs. Alpha-amylase inhibition assay was performed and reported 66% inhibition. In conclusion, currently synthesized Ag2ONPs have exhibited strong biological potential and proved as an attractive eco-friendly candidate. In the future, this preliminary research work will be a helpful source and will open new avenues in diverse fields, including the pharmaceutical, biomedical and pharmacological sectors.

Enhanced Maturation of 3D Bioprinted Skeletal Muscle Tissue Constructs Encapsulating Soluble Factor-Releasing Microparticles.

Several microfabrication technologies have been used to engineer native-like skeletal muscle tissues. However, the successful development of muscle remains a significant challenge in the tissue engineering field. Muscle tissue engineering aims to combine muscle precursor cells aligned within a highly organized 3D structure and biological factors crucial to support cell differentiation and maturation into functional myotubes and myofibers. In this study, the use of 3D bioprinting is proposed for the fabrication of muscle tissues using gelatin methacryloyl (GelMA) incorporating sustained insulin-like growth factor-1 (IGF-1)-releasing microparticles and myoblast cells. This study hypothesizes that functional and mature myotubes will be obtained more efficiently using a bioink that can release IGF-1 sustainably for in vitro muscle engineering. Synthesized microfluidic-assisted polymeric microparticles demonstrate successful adsorption of IGF-1 and sustained release of IGF-1 at physiological pH for at least 21 days. Incorporating the IGF-1-releasing microparticles in the GelMA bioink assisted in promoting the alignment of myoblasts and differentiation into myotubes. Furthermore, the myotubes show spontaneous contraction in the muscle constructs bioprinted with IGF-1-releasing bioink. The proposed bioprinting strategy aims to improve the development of new therapies applied to the regeneration and maturation of muscle tissues.

Brain-on-a-chip: Recent advances in design and techniques for microfluidic models of the brain in health and disease.

Recent advances in biomaterials, microfabrication, microfluidics, and cell biology have led to the development of organ-on-a-chip devices that can reproduce key functions of various organs. Such platforms promise to provide novel insights into various physiological events, including mechanisms of disease, and evaluate the effects of external interventions, such as drug administration. The neuroscience field is expected to benefit greatly from these innovative tools. Conventional ex vivo studies of the nervous system have been limited by the inability of cell culture to adequately mimic in vivo physiology. While animal models can be used, their relevance to human physiology is uncertain and their use is laborious and associated with ethical issues. To date, organ-on-a-chip systems have been developed to model different tissue components of the brain, including brain regions with specific functions and the blood brain barrier, both in normal and pathophysiological conditions. While the field is still in its infancy, it is expected to have major impact on studies of neurophysiology, pathology and neuropharmacology in future. Here, we review advances made and limitations faced in an effort to stimulate development of the next generation of brain-on-a-chip devices.

Investigation of photosensitively bioconjugated targeted quantum dots for the labeling of Cu/Zn superoxide dismutase in fixed cells and tissue sections.

This study presents the development of targeted and antibody cross-linked QDs and explores whether these bioconjugates could specifically and effectively label Cu/Zn superoxide dismutase (SOD1) on fixed cells and tissues. QD-antibody conjugation was achieved by using our previously invented AmiNoacid (monomer) Decorated and Light Underpining Conjugation Approach (ANADOLUCA) method. In this method, we have used a photosensitive aminoacid monomer having ruthenium complex which is a synthetic and inexpensive material for the preparation of bioconjugates. Its specificity was demonstrated by extracting the active enzyme from rat liver lysate by using the bioconjugate. It provided accurate antibody orientation, high specificity and mechanic stability. The protocol steps for QD-antibody conjugation and specimen preparation were described in detail. The nanobioconjugates were prepared under mild conditions (for example in day light), independent of pH and temperature, without affecting conformation and function of protein. This protocol is simple, inexpensive and can be successfully adapted to detect other targets on different cell types and tissues.

Carbonic Anhydrase Carrying Electrospun Nanofibers for Biocatalysis Applications.

BACKGROUND: Enzymes are efficient biocatalysis that catalysis a large number of reactions due to their chemical, regional, or stereo specifities and selectivity. Their usage in bioreactor or biosensor systems has great importance. Carbonic anhydrase enzyme catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. In organisms, the carbonic anhydrase enzyme has crucial roles connected with pH and CO2 homeostasis, respiration, and transport of CO2/bicarbonate, etc. So, immobilization of the enzyme is important in stabilizing the catalyst against thermal and chemical denaturation in bioreactor systems when compared to the free enzyme that is unstable at high temperatures and extreme pH values, as well as in the presence of organic solvents or toxic reagents. Nano-scale composite materials have attracted considerable attention in recent years, and electrospinning based all-nanocomposite materials have a wide range of applications. In this study, electrospun nanofibers were fabricated and used for the supporting media for carbonic anhydrase enzyme immobilization to enhance the enzyme storage and usage facilities. OBJECTIVE: In this article, our motivation is to obtain attractive electrospun support for carbonic anhydrase enzyme immobilization to enhance the enzyme reusability and storage ability in biocatalysis applications. METHODS: In this article, we propose electrospun nanofibers for carbonic anhydrase carrying support for achieving our aforementioned object. In the first part of the study, agar with polyacrylonitrile (PAN) nanofibers was directly fabricated from an agar-PAN mixture solution using the electrospinning method, and fabricated nanofibers were cross-linked via glutaraldehyde (GA). The morphology, chemical structure, and stability of the electrospun nanofibers were characterized. In the second part of the study, the carbonic anhydrase enzyme was immobilized onto fabricated electrospun nanofibers. Then, enzyme activity, the parameters that affect enzyme immobilization such as pH, enzyme amount, immobilization time, etc. and reusability were investigated. RESULTS: When the scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis results are combined in the characterization process of the synthesized electrospun nanofibers, the optimum cross-linking time is found to be 8 hours using 5% glutaraldehyde cross-linking agent. Then, thermal stability measurements showed that the thermal stability of electrospun nanofibers has an excellent characteristic for biomedical applications. The optimum temperature value was found 37°C, pH 8 was determined as an optimum pH, and 100 ppm carbonic anhydrase enzyme concentration was found to be optimum enzyme concentration for the carbonic anhydrase enzyme immobilization. According to the kinetic data, carbonic anhydrase immobilized electrospun nanofibers acted as a biocatalyst in the conversion of the substrate to the product in 83.98%, and immobilized carbonic anhydrase enzyme is reusable up to 9 cycles in biocatalysis applications. CONCLUSION: After applying the framework, we get a new biocatalysis application platform for carbonic anhydrase enzyme. Electrospun nanofibers were chosen as the support material for enzyme immobilization. By using this approach, the carbonic anhydrase enzyme could easily be used in the industrial area by cost-effective advantageous aspects.