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The myotendinous junction (MTJ) is the interface connecting skeletal muscle and tendon tissues. This specialized region represents the bridge that facilitates the transmission of contractile forces from muscle to tendon, and ultimately the skeletal system for the creation of movement. MTJs are, therefore, subject to high stress concentrations, rendering them susceptible to severe, life-altering injuries. Despite the scarcity of knowledge obtained from MTJ formation during embryogenesis, several attempts have been made to engineer this complex interfacial tissue. These attempts, however, fail to achieve the level of maturity and mechanical complexity required for in vivo transplantation. This review summarizes the strategies taken to engineer the MTJ, with an emphasis on how transitioning from static to mechanically inducive dynamic cultures may assist in achieving myotendinous maturity. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman Spectroscopy (SERS) is a powerful optical sensing technique that amplifies the signal generated by Raman scattering by many orders of magnitude. Although the extreme sensitivity of SERS enables an extremely low limit of detection, even down to single molecule levels, it is also a primary limitation of the technique due to its tendency to equally amplify 'noise' generated by non-specifically adsorbed molecules at (or near) SERS-active interfaces. Eliminating interference noise is thus critically important to SERS biosensing and typically involves onerous extraction/purification/washing procedures and/or heavy dilution of biofluid samples. Consequently, direct analysis within biofluid samples or in vivo environments is practically impossible. In this study, an anti-fouling coating of recombinant human Lubricin (LUB) was self-assembled onto AuNP-modified glass slides via a simple drop-casting method. A series of Raman spectra were collected using rhodamine 6G (R6G) as a model analyte, which was spiked into NaCl solution or unprocessed whole blood. Likewise, we demonstrate the same sensing system for the quantitative detection of L -cysteine spiked in undiluted milk. It was demonstrated for the first time that LUB coating can mitigate the deleterious effect of fouling in a SERS sensor without compromising the detection of a target analyte, even in a highly fouling, complex medium like whole blood or milk. This feat is achieved through a molecular sieving property of LUB that separates small analytes from large fouling species directly at the sensing interface resulting in SERS spectra with low background (i.e., noise) levels and excellent analyte spectral fidelity. These findings indicate the great potential for using LUB coatings together with an analyte-selective layer to form a hierarchical separation system for SERS sensing of relevant analytes directly in complex biological media, aquaculture, food matrix or environmental samples. [ABSTRACT FROM AUTHOR]

A major obstacle toward creating human‐scale artificial tissue models is supplying encapsulated cells with oxygen and other nutrients throughout the construct. In particular, creating channels in hydrogels that match the resolution and density of the smallest blood capillaries (≤10 µm) remains highly challenging. Here, a novel method is demonstrated where polycaprolactone fibers printed using melt‐electrowriting are encapsulated in cell‐laden hydrogels and then physically removed to produce hollow, perfusable channels. This technique produces a range of channel diameters (10–41 µm) with circular cross‐sections and in hydrogels representing various crosslinking mechanisms. The channels can be formed as interconnected grids, hierarchically branched patterns, or stacked in layers with ≈200 µm channel spacing, thus matching average capillary density in the human body. Alternatively, selective removal of fibers from a melt electrowriting grid can generate perfusable channels within a reinforcing fiber network. This method can be performed in the presence of cells, with human fibroblasts exhibiting encapsulated in gelatin methacryloyl showing no detectable cytotoxic effects. This technique is a promising approach for creating perfusable channels with very small diameters within cell‐laden hydrogel matrices, with potential applications including in vitro tissue models and hydrogel microfluidics. [ABSTRACT FROM AUTHOR]

The development of electroactive cell-laden hydrogels (bioscaffolds) has gained interest in neural tissue engineering research due to their inherent electrical properties that can induce the regulation of cell behaviour. Hydrogels combined with electrically conducting materials can respond to external applied electric fields, where these stimuli can promote electro-responsive cell growth and proliferation. A successful neural interface for electrical stimulation should present the desired stable electrical properties, such as high conductivity, low impedance, increased charge storage capacity and similar mechanical properties related to a target neural tissue. We report how different electrical stimulation protocols can impact neuronal cells' survival and proliferation when using cell-laden GelMA/GO hydrogels. The rat pheochromocytoma cell line, PC12s encapsulated into hydrogels showed an increased proliferation behaviour with increasing current amplitudes applied. Furthermore, the presence of GO in GelMA hydrogels enhanced the metabolic activity and DNA content of PC12s compared with GelMA alone. Similarly, hydrogels provided survival of encapsulated cells at higher current amplitudes when compared to cells seeded onto ITO flat surfaces, which expressed significant cell death at a current amplitude of 2.50 mA. Our findings provide new rational choices for electroactive hydrogels and electrical stimulation with broad potential applications in neural tissue engineering research. [ABSTRACT FROM AUTHOR]

CDKL5 deficiency disorder (CDD) is an X-linked brain disorder of young children and is caused by pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene. Individuals with CDD suffer infantile onset, drug-resistant seizures, severe neurodevelopmental impairment and profound lifelong disability. The CDKL5 protein is a kinase that regulates key phosphorylation events vital to the development of the complex neuronal network of the brain. Pathogenic variants identified in patients may either result in loss of CDKL5 catalytic activity or are hypomorphic leading to partial loss of function. Whilst the progressive nature of CDD provides an excellent opportunity for disease intervention, we cannot develop effective therapeutics without in-depth knowledge of CDKL5 function in human neurons. In this mini review, we summarize new findings on the function of CDKL5. These include CDKL5 phosphorylation targets and the consequence of disruptions on signaling pathways in the human brain. This new knowledge of CDKL5 biology may be leveraged to advance targeted drug discovery and rapid development of treatments for CDD. Continued development of effective humanized models will further propel our understanding of CDD biology and may permit the development and testing of therapies that will significantly alter CDD disease trajectory in young children. [ABSTRACT FROM AUTHOR]

The debilitating effects of muscle damage, either through ischemic injury or volumetric muscle loss (VML), can have significant impacts on patients, and yet there are few effective treatments. This challenge arises when function is degraded due to significant amounts of skeletal muscle loss, beyond the regenerative ability of endogenous repair mechanisms. Currently available surgical interventions for VML are quite invasive and cannot typically restore function adequately. In response to this, many new bioengineering studies implicate 3D bioprinting as a viable option. Bioprinting for VML repair includes three distinct phases: printing and seeding, growth and maturation, and implantation and application. Although this 3D bioprinting technology has existed for several decades, the advent of more advanced and novel printing techniques has brought us closer to clinical applications. Recent studies have overcome previous limitations in diffusion distance with novel microchannel construct architectures and improved myotubule alignment with highly biomimetic nanostructures. These structures may also enhance angiogenic and nervous ingrowth post-implantation, though further research to improve these parameters has been limited. Inclusion of neural cells has also shown to improve myoblast maturation and development of neuromuscular junctions, bringing us one step closer to functional, implantable skeletal muscle constructs. Given the current state of skeletal muscle 3D bioprinting, the most pressing future avenues of research include furthering our understanding of the physical and biochemical mechanisms of myotube development and expanding our control over macroscopic and microscopic construct structures. Further to this, current investigation needs to be expanded from immunocompromised rodent and murine myoblast models to more clinically applicable human cell lines as we move closer to viable therapeutic implementation. [ABSTRACT FROM AUTHOR]

Extrusion printing techniques are widely used across tissue engineering and related fields for producing 3D structures from biocompatible thermoplastics, however the achievable structural complexity and porosity can be limited by the nozzle‐based, layer‐by‐layer deposition process. Here, how this limitation can be overcome through a new technique termed Negative Embodied Sacrificial Template 3D printing is illustrated. It is demonstrated how the negative pattern within a 3D printed object can easily describe geometries that are extremely challenging to extrusion print directly with biomaterials, and at high resolution. Negative patterns in a water‐soluble sacrificial template can be "developed" by casting in a secondary material and dissolving the template, creating exquisitely complex 3D structures including hyper‐branched dendritic structures and open lattices with stiffnesses tuneable over 3 orders of magnitude. The technique is amenable to a plethora of materials from biodegradable thermoplastics (such as polycaprolactone) to resins (including acrylic and epoxy), silicones (including the Sylgard 184 polydimethylsiloxane formulation), ceramics (including hydroxyapatite composites), hydrogels (including agarose and gelatin methacryloyl), low‐melt temperature metal alloys and others. Using an unmodified, consumer‐grade printer, NEST3D printing achieves high resolution, intricate biomaterial structures with potential applications in biomedical implants and tissue engineering scaffolds. [ABSTRACT FROM AUTHOR]

The ability to prevent or minimize the accumulation of unwanted biological materials on implantable medical devices is important in maintaining the long-term function of implants. To address this issue, there has been a focus on materials, both biological and synthetic, that have the potential to prevent device fouling. In this review, we introduce a glycoprotein called lubricin and report on its emergence as an effective antifouling coating material. We outline the versatility of lubricin coatings on different surfaces, describe the physical properties of its monolayer structures, and highlight its antifouling properties in improving implant compatibility as well as its use in treatment of ocular diseases and arthritis. This review further describes synthetic polymers mimicking the lubricin structure and function. We also discuss the potential future use of lubricin and its synthetic mimetics as antiadhesive biomaterials for therapeutic applications. [ABSTRACT FROM AUTHOR]

Graphene oxide (GO) and other 2D-nanosheet materials exhibit a range of useful physical, chemical, electrical, and optical properties that may usher in the next generation of biomedical, bioimaging, or sensing technologies. One limitation of GO is its poor stability in concentrated electrolytes and other complex fluids that requires steric stabilizers, including surface grafted polymers, to overcome attractive van der Waals interactions. Here, we describe a simple, rapid, and highly effective method of modifying GO and other 2D-nanosheets with thick, grafted (bio)polymer brushes via the solution self-assembly of lubricin (LUB; a.k.a. PRG4), an antiadhesive glycoprotein. Atomic force microscopy (AFM) imaging and force measurements were used to characterize the morphology and nanomechanical response of these LUB–GO, 2D-nanosheet complexes (2D-NSC). These characterization studies reveal a strong correlation between the GO surface area and the thickness (i.e., molecular extension) of the grafted LUB brush caused by edge free volume effects. Likewise, this edge free volume influences the extension of the LUB brush structure more than 300 nm away from the edge, resulting in a transition region of increasing brush extension before reaching a fully extended state within the central regions of the 2D-NSC. Fitting AFM normal force measurements using an adapted Alexander–de Gennes polymer brush model also indicate that the edge free volume leads to a mechanical softening of the LUB brush due to the lateral spreading and/or deflection of LUB molecules under compression. Finally, the stability studies of 2D-NSCs dispersed in concentrated electrolyte solutions demonstrate the effectiveness of the grafted LUB brushes at inhibiting aggregation even in the harshest environments. 2D-NSCs thus represent a simple solution to modifying nanosheets with thick, multifunctional brushes with promising application in biosensing, bioimaging, catalysis, and biolubrication, where nanosheets must perform in concentrated electrolytes or complex fluids. [ABSTRACT FROM AUTHOR]

The electrochemical sensing of clonazepam in samples of unprocessed saliva is reported, using commercially available reduced graphene oxide (rGO) screen printed electrodes (SPEs). The rGO electrodes were modified with self‐assembled lubricin (LUB) antiadhesive coatings that function as a size‐selective molecular sieve that partitions small molecule analytes from large fouling molecules at the electrode‐fluid interface. By mitigating the adverse effects of protein fouling, these LUB coatings enable direct, reliable, and quantitative electrochemical measurements of clonazepam concentrations in untreated saliva, which was not possible with uncoated rGO electrodes in identical tests. The proposed assay design, which is suitable for disposable point‐of‐care diagnostics requires no sample pre‐treatment and shows a linear detection response over a concentration range of 25–250 nM with a correlation coefficient of R2=0.987 and a limit of detection (LoD) of 16.67 nM. The swab‐based sample acquisition method paired with the antifouling properties of the LUB coatings provide a cost effective and practical biosensing platform for assessing electroactive drug concentrations in saliva in a point‐of‐care setting. [ABSTRACT FROM AUTHOR]

Biofouling on surfaces compromises the function of biomedical devices whose function involves contact with biological fluids. In the context of electrochemical devices, proteins are attracted to the surface via coaction of various forces (hydrogen bond, hydrophobic effect, and other polar interactions) and protein interaction with the surface can significantly alter the surface chemistry. In response to this issue, we have developed an efficient anti‐biofouling surface that employs a glycoprotein, lubricin (LUB), and which generates low impedance layers compatible with electrochemical applications. Herein, we investigate how different LUB densities on platinum (Pt) electrodes affect the surface electrochemistry and its ability to prevent nonspecific adsorption of protein to the surface. Surfaces with higher densities of LUB were more resistant to protein adsorption. The LUB modified Pt electrodes were challenged in artificial perilymph (AP) media under passive and electrically stimulated conditions over 7‐day periods throughout which the LUB layer retained its anti‐biofouling and surface coating stability. LUBed up: a low‐impedance protein‐resistant surface is introduced, that consists of lubricin (LUB) coating onto a platinum surface. The LUB‐platinum interface presents a good resistance to nonspecific protein adsorption, remaining stable on the surface under passive or electrical‐stimulated conditions. [ABSTRACT FROM AUTHOR]

Trilayered polypyrrole (PPy) actuators have high stress density, low modulus and have wide potential biological applications including use in artificial muscles and in limb prosthesis after limb amputation. This article examines the in vivo biocompatibility of actuators in muscle using rabbit models. The actuators were specially designed with pores to encourage tissue in growth; this study also assessed the effect of such pores on the stability of the actuators in vivo. Trilayered PPy actuators were either laser cut with 150 µm pores or left pore-less and implanted into rabbit muscle for 3 days, 2 weeks, 4 weeks and 8 weeks and retrieved subsequently for histological analysis. In a second set of experiments, the cut edges of pores in porous actuator strips were further sealed by PPy after laser cutting to further improve its stability in vivo. Porous actuators with and without PPy sealing of pore edges were implanted intramuscularly for 4 and 8 weeks and assessed with histology. Pore-less actuators incited a mild inflammatory response, becoming progressively walled off by a thin layer of fibrous tissue. Porous actuators showed increased PPy fragmentation and delamination with associated greater foreign body response compared to pore-less actuators. The PPy fragmentation was minimized when the pore edges were sealed off by PPy after laser cutting showing less PPy debris. Laser cutting of the actuators with pores destabilizes the PPy. This can be overcome by sealing the cut edges of the pores with PPy after laser. The findings in this article have implications in future design and manufacturing of PPy actuator for use in vivo. Graphical abstract: [ABSTRACT FROM AUTHOR]

In the current study, we describe the synthesis, material characteristics, and cytocompatibility of conducting poly (ɛ-caprolactone) (PCL)-based nano-composite films. Electrically conducting carbon nano-fillers (carbon nano-fiber (CNF), nano-graphite (NG), and liquid exfoliated graphite (G)) were used to prepare porous film type scaffolds using modified solvent casting methods. The electrical conductivity of the nano-composite films was increased when carbon nano-fillers were incorporated in the PCL matrix. CNF-based nano-composite films showed the highest increase in electrical conductivity. The presence of an ionic solution significantly improved the conductivity of some of the polymers, however at least 24 h was required to absorb the simulated ion solutions. CNF-based nano-composite films were found to have good thermo-mechanical properties compared to other conducting polymer films due to better dispersion and alignment in the critical direction. Increased nano-filler content increased the crystallisation temperature. Analysis of cell viability revealed no increase in cell death on any of the polymers compared to tissue culture plastic controls, or compared to PCL polymer without nano-composites. The scaffolds showed some variation when tested for PC12 cell attachment and proliferation, however all the polymers supported PC12 attachment and differentiation in the absence of cell adhesion molecules. In general, CNF-based nano-composite films with highest electrical conductivity and moderate roughness showed highest cell attachment and proliferation. These polymers are promising candidates for use in neural applications in the area of bionics and tissue engineering due to their unique properties. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 853-865, 2016. [ABSTRACT FROM AUTHOR]

Introduction: Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. Methods: Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. Results: Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. Conclusion: Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery. [ABSTRACT FROM AUTHOR]

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a ‘cap’ on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft. [ABSTRACT FROM AUTHOR]