Deoxyribonucleic acid (DNA) emerges as building bricks for the fabrication of nanostructure with complete artificial architecture and geometry. The amazing ability of DNA in building two- and three-dimensional structures raises the possibility of developing smart nanomachines with versatile controllability for various applications. Here, we overviewed the recent progresses in engineering DNA machines for specific bioengineering and biomedical applications.
Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future.
Lateral flow assays (LFAs) hold great promise for point-of-care testing, especially in resource-poor settings. However, the poor sensitivity of LFAs limits their widespread applications. To address this, we developed a novel device by integrating dialysis-based concentration method into LFAs. The device successfully achieved 10-fold signal enhancement in Human Immunodeficiency Virus (HIV) nucleic acid detection with a detection limit of 0.1nM and 4-fold signal enhancement in myoglobin (MYO) detection with a detection limit of 1.56ng/mL in less than 25min. This simple, low-cost and portable integrated device holds great potential for highly sensitive detection of various target analytes for medical diagnostics, food safety analysis and environmental monitoring.
The identification and quantification of chemicals play a vital role in evaluation and surveillance of environmental health and safety. However, current techniques usually depend on costly equipment, professional staff, and/or essential infrastructure, limiting their accessibility. In this work, we develop paper-based capacitive sensors (PCSs) that allow simple, rapid identification and quantification of various chemicals from microliter size samples with the aid of a handheld multimeter. PCSs are low-cost parallel-plate capacitors (~$0.01 per sensor) assembled from layers of aluminum foil and filter paper via double-sided tape. The developed PCSs can identify different kinds of fluids (e.g., organic chemicals) and quantify diverse concentrations of substances (e.g., heavy metal ions) based on differences in dielectric properties, including capacitance, frequency spectrum, and dielectric loss tangent. The PCS-based method enables chemical identification and quantification to take place much cheaply, simply, and quickly at the point-of-care (POC), holding great promise for environmental monitoring in resource-limited settings.
It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits' (α, β, γ, and δ) expression was observed in hMSCs, although only α subunit was significantly increased during stretching. An increase in tenogenic genes' (collagen1, collagen3, decorin, tenascin-c, scleraxis, and tenomodulin) and proteins' (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.
The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.
In this article, a combination of far field electrospinning (FFES) and free-radical polymerization has been used to create a unique platform for protein immobilization via the physical attachment of biomolecules to the surface of the fiber mats. The large specific surface area of the fibers with its tailored chemistry provides a desirable platform for effective analyte-surface interaction. The detailed analysis of protein immobilization on a newly developed bio-receptive surface plays a vital role to gauge its advantages in bio-diagnostic applications. We relied on scanning electron microscopy (SEM), diameter range analysis, and X-ray photoelectron spectroscopy (XPS), along with thermal gravimetric analysis (TGA), water-in-air contact angle analysis (WCA), Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) to study our developed platforms and to provide valuable information regarding the presence of biomolecular entities on the surface. Detailed analyses of the fiber mats before and after antibody immobilization have shown obvious changes on the surface of the bioreceptive surface including: (i) an additional peak corresponding to the presence of an antibody in TGA analysis; (ii) extra FTIR peaks corresponding to the presence of antibodies on the coated fiber platforms; and (iii) a clear alteration in surface roughness recorded by AFM analysis. Confirmation analyses on protein immobilization are of great importance as they underlay substantial grounds for various biosensing applications.
Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems.
The complex architecture of the cortical part of the bovine-femur was examined to develop potential tissue engineering (TE) scaffolds. Weight-change and X-ray diffraction (XRD) results show that significant phase transformation and morphology conversion of the bone occur at 500-750°C and 750-900°C, respectively. Another breakthrough finding was achieved by determining a sintering condition for the nucleation of hydroxyapatite crystal from bovine bone via XRD technique. Scanning electron microscopy results of morphological growth suggests that the concentration of polymer fibrils increases (or decreases, in case of apatite crystals) from the distal to proximal end of the femur. Energy-dispersive analysis of X-ray, Fourier transform infrared, micro-computer tomography, and mechanical studies of the actual composition also strongly support our microscopic results and firmly indicate the functionally graded material properties of bovine-femur. Bones sintered at 900 and 1000°C show potential properties for soft and hard TE applications, respectively.
Scaffold design from xenogeneic bone has the potential for tissue engineering (TE). However, major difficulties impede this potential, such as the wide range of properties in natural bone. In this study, sintered cortical bones from different parts of a bovine-femur impregnated with biodegradable poly(ethylene glycol) (PEG) binder by liquid phase adsorption were investigated. Flexural mechanical properties of the PEG-treated scaffolds showed that the scaffold is stiffer and stronger at a sintering condition of 1000°C compared with 900°C. In vitro cytotoxicity of the scaffolds evaluated by Alamar Blue assay and microscopic tests on human fibroblast cells is better at 1000°C compared with that at 900°C. Furthermore, in vitro biocompatibility and flexural property of scaffolds derived from different parts of a femur depend on morphology and heat-treatment condition. Therefore, the fabricated scaffolds from the distal and proximal parts at 1000°C are potential candidates for hard and soft TE applications, respectively.
Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.
Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future.
One of the major challenges in bone grafting is the lack of sufficient bone vascularization. A rapid and stable bone vascularization at an early stage of implantation is essential for optimal functioning of the bone graft. To address this, the ability of in situ TiO2 nanofibrous surfaces fabricated via thermal oxidation method to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) was investigated. The cellular responses of HUVECs on TiO2 nanofibrous surfaces were studied through cell adhesion, cell proliferation, capillary-like tube formation, growth factors secretion (VEGF and BFGF), and angiogenic-endogenic-associated gene (VEGF, VEGFR2, BFGF, PGF, HGF, Ang-1, VWF, PECAM-1 and ENOS) expression analysis after 2 weeks of cell seeding. Our results show that TiO2 nanofibrous surfaces significantly enhanced adhesion, proliferation, formation of capillary-like tube networks and growth factors secretion of HUVECs, as well as leading to higher expression level of all angiogenic-endogenic-associated genes, in comparison to unmodified control surfaces. These beneficial effects suggest the potential use of such surface nanostructures to be utilized as an advantageous interface for bone grafts as they can promote angiogenesis, which improves bone vascularization.
The concentration of biological components of synovial fluid (such as albumin, globulin, hyaluronic acid, and lubricin) varies between healthy persons and osteoarthritis (OA) patients. The aim of the present study is to compare the effects of such variation on tribological performance in a simulated hip joint model. The study was carried out experimentally by utilizing a pin-on-disk simulator on ceramic-on-ceramic (CoC) and ceramic-on-polyethylene (CoP) hip joint implants. The experimental results show that both friction and wear of artificial joints fluctuate with the concentration level of biological components. Moreover, the performance also varies between material combinations. Wear debris sizes and shapes produced by ceramic and polyethylene were diverse. We conclude that the biological components of synovial fluid and their concentrations should be considered in order to select an artificial hip joint to best suit that patient.
Osteoarthritis-oriented synovial fluid (OASF), i.e., that typical of a patient with osteoarthritis, has different physical and biological characteristics than bovine serum (BS), a lubricant widely used in biotribological investigations. Micro-dimpled and diamond-like carbon- (DLC) coated surfaces are key emerging interfaces for orthopedic implants. In this study, tribological performances of dimpled surfaces, with and without DLC coating, have been investigated under both BS and OASF. The friction tests were performed utilizing a pin on a disk tribometer, whereas contact pressure, speed, and temperature were simulated to a 'medium walking gait' of hip joint conditions. The mechanical properties of the specimen and the physical properties of the lubricant were characterized before the friction test. Raman analysis was conducted to identify the coating condition both before and after the test. The DLC-coated dimpled surface showed maximum hardness and residual stress. A DLC-coated dimpled surface under an OASF lubricated condition yielded a lower friction coefficient and wear compared to those of plain and dimpled specimens. The higher graphitization of coated materials with increasing load was confirmed by Raman spectroscopy.
Native cartilage matrix derived (CMD) scaffolds from various animal and human sources have drawn attention in cartilage tissue engineering due to the demonstrable presence of bioactive components. Different chemical and physical treatments have been employed to enhance the micro-architecture of CMD scaffolds. In this study we have assessed the typical effects of physical cross-linking methods, namely ultraviolet (UV) light, dehydrothermal (DHT) treatment, and combinations of them on bovine articular CMD porous scaffolds with three different matrix concentrations (5%, 15% and 30%) to assess the relative strengths of each treatment. Our findings suggest that UV and UV-DHT treatments on 15% CMD scaffolds can yield architecturally optimal scaffolds for cartilage tissue engineering.
There has been unprecedented development in tissue engineering (TE) over the last few years owing to its potential applications, particularly in bone reconstruction or regeneration. In this article, we illustrate several advantages and disadvantages of different approaches to the design of electrospun TE scaffolds. We also review the major benefits of electrospun fibers for three-dimensional scaffolds in hard connective TE applications and identify the key strategies that can improve the mechanical properties of scaffolds for bone TE applications. A few interesting results of recent investigations have been explained for future trends in TE scaffold research.
To understand how cells respond to the nanoscale extracellular environment in vivo, cells from various sources have been cultured on nanoscale patterns fabricated using bottom-up and top-down techniques. Human fetal osteoblasts (hFOBs) and stem cells are some of them and they are known to be overtly responsive to nanoscale topographies - allowing us to investigate the hows and whys of the response in vitro. Information gathered from these in vitro studies could be used to control the cells, i.e. make the stem cells differentiate or retain their characteristics without the use of medium supplements. In this review, hFOB and stem cell responses to nanotopographies are summarized and discussed to shed some light on the influence of patterns on the reactions. Although both types of cells are responsive to nanoscale topographies, the responses are found to be unique to topographical dimension, shape, orientation and the types of cells used. This implies that cellular responses are influenced by multitude of factors and that if done right, cheaper self-assembled nanotopographies can be tailored to control the cells. A new self-assembly, powder-based technique is also included to provide an insight into the future of nanofabrication.
(1) Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCL-HA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation.
Electrospun polycaprolactone (PCL) nanofibers have emerged as a promising material in diverse biomedical applications due to their various favorable features. However, their application in the field of biosensors such as point-of-care lateral flow assays (LFA) has not been investigated. The present study demonstrates the use of electrospun PCL nanofibers as a reaction membrane for LFA. Electrospun PCL nanofibers were treated with NaOH solution for different concentrations and durations to achieve a desirable flow rate and optimum detection sensitivity in nucleic acid-based LFA. It was observed that the concentration of NaOH does not affect the physical properties of nanofibers, including average fiber diameter, average pore size and porosity. However, interestingly, a significant reduction of the water contact angle was observed due to the generation of hydroxyl and carboxyl groups on the nanofibers, which increased their hydrophilicity. The optimally treated nanofibers were able to detect synthetic Zika viral DNA (as a model analyte) sensitively with a detection limit of 0.5 nM. Collectively, the benefits such as low-cost of fabrication, ease of modification, porous nanofibrous structures and tunability of flow rate make PCL nanofibers a versatile alternative to nitrocellulose membrane in LFA applications. This material offers tremendous potential for a broad range of point-of-care applications.