Dependence of crack shape in loaded articular cartilage on the collagenous structure.

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages. METHODS: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance. RESULTS: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues. CONCLUSION: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.

Paper-based point-of-care testing for diagnosis of dengue infections.

Dengue endemic is a serious healthcare concern in tropical and subtropical countries. Although well-established laboratory tests can provide early diagnosis of acute dengue infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Point-of-care (POC) devices, particularly paper-based POC devices, are typically rapid, cost-effective and user-friendly, and they can be used as diagnostic tools for the prompt diagnosis of dengue at POC settings. Here, we review the importance of rapid dengue diagnosis, current dengue diagnostic methods, and the development of paper-based POC devices for diagnosis of dengue infections at the POC.

Advances in paper-based sample pretreatment for point-of-care testing.

In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.

Hypoxia enhances the viability, growth and chondrogenic potential of cryopreserved human adipose-derived stem cells.

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O2) and 2) hypoxia (2% O2) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.

Polymethacrylate Coated Electrospun PHB Fibers as a Functionalized Platform for Bio-Diagnostics: Confirmation Analysis on the Presence of Immobilized IgG Antibodies against Dengue Virus.

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.

Polydimethylsiloxane-Paper Hybrid Lateral Flow Assay for Highly Sensitive Point-of-Care Nucleic Acid Testing.

In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future.

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

Hydrogel-based methods for engineering cellular microenvironment with spatiotemporal gradients.

Natural cellular microenvironment consists of spatiotemporal gradients of multiple physical (e.g. extracellular matrix stiffness, porosity and stress/strain) and chemical cues (e.g. morphogens), which play important roles in regulating cell behaviors including spreading, proliferation, migration, differentiation and apoptosis, especially for pathological processes such as tumor formation and progression. Therefore, it is essential to engineer cellular gradient microenvironment incorporating various gradients for the fabrication of normal and pathological tissue models in vitro. In this article, we firstly review the development of engineering cellular physical and chemical gradients with cytocompatible hydrogels in both two-dimension and three-dimension formats. We then present current advances in the application of engineered gradient microenvironments for the fabrication of disease models in vitro. Finally, concluding remarks and future perspectives for engineering cellular gradients are given.

An integrated lateral flow assay for effective DNA amplification and detection at the point of care.

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.

Antibacterial properties of poly (octanediol citrate)/gallium-containing bioglass composite scaffolds.

Bioactive glasses may function as antimicrobial delivery systems through the incorporation and subsequent release of therapeutic ions. The aim of this study was to evaluate the antimicrobial properties of a series of composite scaffolds composed of poly(octanediol citrate) with increased loads of a bioactive glass that releases zinc (Zn(2+)) and gallium (Ga(3+)) ions in a controlled manner. The antibacterial activity of these scaffolds was investigated against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The ability of the scaffolds to release ions and the subsequent ingress of these ions into hard tissue was evaluated using a bovine bone model. Scaffolds containing bioactive glass exhibited antibacterial activity and this increased in vitro with higher bioactive glass loads; viable cells decreased to about 20 % for the composite scaffold containing 30 % bioactive glass. The Ga(3+) release rate increased as a function of time and Zn(2+) was shown to incorporate into the surrounding bone.

Osteoblast and stem cell response to nanoscale topographies: a review.

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.

Mechanoregulation of cardiac myofibroblast differentiation: implications for cardiac fibrosis and therapy.

Cardiac myofibroblast differentiation, as one of the most important cellular responses to heart injury, plays a critical role in cardiac remodeling and failure. While biochemical cues for this have been extensively investigated, the role of mechanical cues, e.g., extracellular matrix stiffness and mechanical strain, has also been found to mediate cardiac myofibroblast differentiation. Cardiac fibroblasts in vivo are typically subjected to a specific spatiotemporally changed mechanical microenvironment. When exposed to abnormal mechanical conditions (e.g., increased extracellular matrix stiffness or strain), cardiac fibroblasts can undergo myofibroblast differentiation. To date, the impact of mechanical cues on cardiac myofibroblast differentiation has been studied both in vitro and in vivo. Most of the related in vitro research into this has been mainly undertaken in two-dimensional cell culture systems, although a few three-dimensional studies that exist revealed an important role of dimensionality. However, despite remarkable advances, the comprehensive mechanisms for mechanoregulation of cardiac myofibroblast differentiation remain elusive. In this review, we introduce important parameters for evaluating cardiac myofibroblast differentiation and then discuss the development of both in vitro (two and three dimensional) and in vivo studies on mechanoregulation of cardiac myofibroblast differentiation. An understanding of the development of cardiac myofibroblast differentiation in response to changing mechanical microenvironment will underlie potential targets for future therapy of cardiac fibrosis and failure.

Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1ß in cartilage explants.

C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). SEM showed uniform, 2 to 3 µm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1ß and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1ß in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

Direct writing electrodes using a ball pen for paper-based point-of-care testing.

The integration of paper with an electrochemical device has attracted growing attention for point-of-care testing, where it is of great importance to fabricate electrodes on paper in a low-cost, easy and versatile way. In this work, we report a simple strategy for directly writing electrodes on paper using a pressure-assisted ball pen to form a paper-based electrochemical device (PED). This method is demonstrated to be capable of fabricating electrodes on paper with good electrical conductivity and electrochemical performance, holding great potential to be employed in point-of-care applications, such as in human health diagnostics and food safety detection. As examples, the PEDs fabricated using the developed method are applied for detection of glucose in artificial urine and melamine in sample solutions. Furthermore, our developed strategy is also extended to fabricate PEDs with multi-electrode arrays and write electrodes on non-planar surfaces (e.g., paper cup, human skin), indicating the potential application of our method in other fields, such as fabricating biosensors, paper electronics etc.

Tribological investigation of diamond-like carbon coated micro-dimpled surface under bovine serum and osteoarthritis oriented synovial fluid.

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.

Tribological performance of the biological components of synovial fluid in artificial joint implants.

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.

Impact of low oxygen tension on stemness, proliferation and differentiation potential of human adipose-derived stem cells.

Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O2 tension on their functional properties has not been well determined. In this study, we investigated the effects of O2 tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O2) and hypoxia (2% O2). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O2 tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.

New method for the isolation of endothelial cells from large vessels.

BACKGROUND AIMS: Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers. METHODS: With the use of this method, the lumen of a bovine aorta was filled with wash medium and the outer surface of the sample was washed with alcohol for 30 seconds. Under a laminar flow hood, the inner surface of the sample was covered with filter paper. Collagenase type II was dripped onto the filter paper as a digestion enzyme. The digestion fluid was seeded in T25 flasks and fed with complete medium every 3 days. RESULTS: The isolated cells were characterized by markers such as CD31, von Willebrand factor, 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate acetylated low-density lipoprotein and angiogenesis behavior. The purity of endothelial cells was detected by flow cytometry to be of nearly 90% purity; these results were confirmed by immunostaining. Moreover, endothelial cells formed blood vessel-like tubes in a three-dimensional environment, which is specific dynamic behavior for endothelial cells. CONCLUSIONS: The new strategy presented in the current report enables isolation of a highly pure population of endothelial cells that can survive long-term culture without inducing an overgrowth of fibroblast cells.

A comparison study of different physical treatments on cartilage matrix derived porous scaffolds for tissue engineering applications.

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.

The properties of chondrocyte membrane reservoirs and their role in impact-induced cell death.

Impact loading of articular cartilage causes extensive chondrocyte death. Cell membranes have a limited elastic range of 3-4% strain but are protected from direct stretch during physiological loading by their membrane reservoir, an intricate pattern of membrane folds. Using a finite-element model, we suggested previously that access to the membrane reservoir is strain-rate-dependent and that during impact loading, the accessible membrane reservoir is drastically decreased, so that strains applied to chondrocytes are directly transferred to cell membranes, which fail when strains exceed 3-4%. However, experimental support for this proposal is lacking. The purpose of this study was to measure the accessible membrane reservoir size for different membrane strain rates using membrane tethering techniques with atomic force microscopy. We conducted atomic force spectroscopy on isolated chondrocytes (n = 87). A micron-sized cantilever was used to extract membrane tethers from cell surfaces at constant pulling rates. Membrane tethers could be identified as force plateaus in the resulting force-displacement curves. Six pulling rates were tested (1, 5, 10, 20, 40, and 80 µm/s). The size of the membrane reservoir, represented by the membrane tether surface areas, decreased exponentially with increasing pulling rates. The current results support our theoretical findings that chondrocytes exposed to impact loading die because of membrane ruptures caused by high tensile membrane strain rates.