Structural Cues Research Articles

RAP2 mediates mechanoresponses of the Hippo pathway.

Mammalian cells are surrounded by neighboring cells and extracellular matrix (ECM), which provide cells with structural support and mechanical cues that influence diverse biological processes1. The Hippo pathway effectors YAP and TAZ are regulated by mechanical cues and mediate cellular responses to ECM stiffness2,3. Here we identified Ras-related GTPase RAP2 as a critical intracellular signal transducer that relays ECM rigidity signals to control mechano-sensitive cellular activities through YAP/TAZ. RAP2 is activated by low ECM stiffness, and RAP2 deletion blocks YAP/TAZ regulation by stiffness signals and promotes aberrant cell growth. Mechanistically, matrix stiffness acts through phospholipase Cγ1 (PLCγ1) to influence levels of phosphatidylinositol 4,5-bisphosphate (PIP2) and its product phosphatidic acid (PA), which activates RAP2 through PDZGEF1/2. At low stiffness, active RAP2 binds to and stimulates mitogen-activated protein kinase kinase kinase kinase 4/6/7 (MAP4K4/6/7) and Rho GTPase activating protein 29 (ARHGAP29), resulting in LATS1/2 activation and YAP/TAZ inhibition. RAP2 and YAP/TAZ play pivotal roles in mechano-regulated transcription, as YAP/TAZ deletion abolishes the ECM stiffness-responsive transcriptome. Our findings reveal RAP2 as a molecular switch in mechanotransduction, thereby defining a mechanosignaling pathway from ECM stiffness to the nucleus.

Hyperekplexia-associated mutations in the neuronal glycine transporter 2

Hyperekplexia or startle disease is a dysfunction of inhibitory glycinergic neurotransmission characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. Although rare, this disorder can have serious consequences, including sudden infant death. One of the most frequent causes of hyperekplexia are mutations in the SLC6A5 gene, encoding the neuronal glycine transporter 2 (GlyT2), a key component of inhibitory glycinergic presynapses involved in synaptic glycine recycling though sodium and chloride-dependent co-transport. Most GlyT2 mutations detected so far are recessive, but two dominant missense mutations have been described. The detailed analysis of these mutations has revealed structural cues on the quaternary structure of GlyT2, and opens the possibility that novel selective pharmacochaperones have potential therapeutic effects in hyperekplexia.

TiO₂ Nanotubes Enhance Vascularization and Osteogenic Differentiation Through Stimulating Interactions Between Bone Marrow Stromal Cells and Endothelial Cells.

The effects of titanium dioxide nanotubes (TNs) on bone repair through affecting the behaviors of mesenchymal stem cells and endothelial cells have been extensively investigated. However, their effects on cell-cell interactions have rarely been reported. Herein, the structural effects (especially the nanotube diameter) of TNs on the cellular behaviors and cell-cell interaction of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) were investigated. Results demonstrated that, as compared to pure Ti foil, the TNs substrates could significantly improve cell proliferation of HBMSCs and HUVECs. In addition, co-cultured HBMSCs and HUVECs showed better adhesion and spreading than mono-cultured cells when they were cultured on the same substrates. Further investigation on co-cultures suggested that the structural cues of TNs could effectively enhance the cell-cell interactions of co-cultured HBMSCs-HVUECs by stimulating cell-cell communications through cell junctions and paracrine effects, which subsequently upregulated the expression of typical osteogenic differentiation genes and vascularization genes and accelerated the vascularization of co-cultured HUVECs and osteogenic differentiation of co-cultured HBMSCs. Besides, TNs with diameter of 100 nm showed preferable stimulatory effects on cell proliferation and differentiation than TNs with diameter of 30 nm. Taken together, structural cues of TNs can significantly affect cell-cell interactions and it can be anticipated that vascularized bone tissues can be constructed by regulating the cell-cell interactions of osteogenic cells and endothelial cells with optimized TNs structural cues, which could eventually result in the enhancement of bone healing in vivo.

Dual Coding of Frequency Modulation in the Ventral Cochlear Nucleus.

Frequency modulation (FM) is a common acoustic feature of natural sounds and is known to play a role in robust sound source recognition. Auditory neurons show precise stimulus-synchronized discharge patterns that may be used for the representation of low-rate FM. However, it remains unclear whether this representation is based on synchronization to slow temporal envelope (ENV) cues resulting from cochlear filtering or phase locking to faster temporal fine structure (TFS) cues. To investigate the plausibility of those encoding schemes, single units of the ventral cochlear nucleus of guinea pigs of either sex were recorded in response to sine FM tones centered at the unit's best frequency (BF). The results show that, in contrast to high-BF units, for modulation depths within the receptive field, low-BF units (<4 kHz) demonstrate good phase locking to TFS. For modulation depths extending beyond the receptive field, the discharge patterns follow the ENV and fluctuate at the modulation rate. The receptive field proved to be a good predictor of the ENV responses for most primary-like and chopper units. The current in vivo data also reveal a high level of diversity in responses across unit types. TFS cues are mainly conveyed by low-frequency and primary-like units and ENV cues by chopper and onset units. The diversity of responses exhibited by cochlear nucleus neurons provides a neural basis for a dual-coding scheme of FM in the brainstem based on both ENV and TFS cues.SIGNIFICANCE STATEMENT Natural sounds, including speech, convey informative temporal modulations in frequency. Understanding how the auditory system represents those frequency modulations (FM) has important implications as robust sound source recognition depends crucially on the reception of low-rate FM cues. Here, we recorded 115 single-unit responses from the ventral cochlear nucleus in response to FM and provide the first physiological evidence of a dual-coding mechanism of FM via synchronization to temporal envelope cues and phase locking to temporal fine structure cues. We also demonstrate a diversity of neural responses with different coding specializations. These results support the dual-coding scheme proposed by psychophysicists to account for FM sensitivity in humans and provide new insights on how this might be implemented in the early stages of the auditory pathway.

Adaptation to Noise in Human Speech Recognition Unrelated to the Medial Olivocochlear Reflex.

Sensory systems constantly adapt their responses to the current environment. In hearing, adaptation may facilitate communication in noisy settings, a benefit frequently (but controversially) attributed to the medial olivocochlear reflex (MOCR) enhancing the neural representation of speech. Here, we show that human listeners (N = 14; five male) recognize more words presented monaurally in ipsilateral, contralateral, and bilateral noise when they are given some time to adapt to the noise. This finding challenges models and theories that claim that speech intelligibility in noise is invariant over time. In addition, we show that this adaptation to the noise occurs also for words processed to maintain the slow-amplitude modulations in speech (the envelope) disregarding the faster fluctuations (the temporal fine structure). This demonstrates that noise adaptation reflects an enhancement of amplitude modulation speech cues and is unaffected by temporal fine structure cues. Last, we show that cochlear implant users (N = 7; four male) show normal monaural adaptation to ipsilateral noise. Because the electrical stimulation delivered by cochlear implants is independent from the MOCR, this demonstrates that noise adaptation does not require the MOCR. We argue that noise adaptation probably reflects adaptation of the dynamic range of auditory neurons to the noise level statistics.SIGNIFICANCE STATEMENT People find it easier to understand speech in noisy environments when they are given some time to adapt to the noise. This benefit is frequently but controversially attributed to the medial olivocochlear efferent reflex enhancing the representation of speech cues in the auditory nerve. Here, we show that the adaptation to noise reflects an enhancement of the slow fluctuations in amplitude over time that are present in speech. In addition, we show that adaptation to noise for cochlear implant users is not statistically different from that for listeners with normal hearing. Because the electrical stimulation delivered by cochlear implants is independent from the medial olivocochlear efferent reflex, this demonstrates that adaptation to noise does not require this reflex.

Heparin functionalization increases retention of TGF-β2 and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering

The tendon/ligament-to-bone transition (enthesis) is a highly specialized interphase tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interphases such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor β2 (TGF-β2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-β2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-β2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-β2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration. Statement of significanceRegeneration of the tendon/ligament-to-bone interphase (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interphases.

Restoring binaural and spatial hearing in cochlear implant users

Adults and children who receive bilateral cochlear implants (BiCIs) have the potential to benefit from the integration of inputs arriving at the brain from both ears. Several factors play a key role in determining if patients will demonstrate binaural sensitivity. We are exploring these factors using two experimental approaches. In the first approach, BiCI users receive pulsatile stimulation to specific pairs of electrodes using research processors that synchronize stimulation with fidelity. We vary stimulus parameters such as temporal fine structure and envelope cues, places of stimulation along the cochleae, and number of electrodes to find parameters that maximize sensitivity to interaural differences for each patient. We also investigate the role of the electrode-neuron interface which is affected by numerous factors including neural health. In a second stimulation approach, we use clinical speech processor to deliver binaural stimulation designed specifically for that patient based on their clinical MAP. In these studies, we are using both standard psychophysics and eye gaze paradigms to understand the underlying processing involved in binaural and spatial hearing. This combined approach is enabling us to design multi-channel multi-rate stimulation strategies aimed at restoring binaural sensitivity and preserving speech understanding.Adults and children who receive bilateral cochlear implants (BiCIs) have the potential to benefit from the integration of inputs arriving at the brain from both ears. Several factors play a key role in determining if patients will demonstrate binaural sensitivity. We are exploring these factors using two experimental approaches. In the first approach, BiCI users receive pulsatile stimulation to specific pairs of electrodes using research processors that synchronize stimulation with fidelity. We vary stimulus parameters such as temporal fine structure and envelope cues, places of stimulation along the cochleae, and number of electrodes to find parameters that maximize sensitivity to interaural differences for each patient. We also investigate the role of the electrode-neuron interface which is affected by numerous factors including neural health. In a second stimulation approach, we use clinical speech processor to deliver binaural stimulation designed specifically for that patient based on their clinical ...

Difficulty with understanding speech in background noise is predicted by broad binaural pitch fusion in bimodal cochlear implant users

Combined use of a cochlear implant (CI) and a contralateral hearing aid has been shown to improve CI users’ speech perception performance in multi-talker environments, presumably due to the use of acoustic temporal fine structure cues for separating one talker from several other talkers. However, there is large variability in this bimodal benefit. In this study, we show that differences in width of binaural pitch fusion, the fusion of dichotically presented tones that evoke different pitches across ears, explain a large part of this variability. Specifically, broad binaural pitch fusion could lead to fusion of multiple voices as one voice, and reduce the ability to use voice pitch differences to separate voices and understand speech in a multi-talker environment. Speech reception thresholds measured using male and female target talkers were compared with binaural pitch fusion results. Overall performance improved with different genders for target and maskers. A strong negative correlation was observed between voice gender benefit and breadth of binaural pitch fusion. These results suggest that sharp binaural pitch fusion is necessary for maximal speech perception in noise when acoustic hearing is available to transmit voice pitch cues. [Work supported by NIH-NIDCD Grant Nos. R01 DC01337 and F32 DC016193.]

Harnessing biochemical and structural cues for tenogenic differentiation of adipose derived stem cells (ADSCs) and development of an in vitro tissue interface mimicking tendon-bone insertion graft

Tendon-bone interface tissue is extremely challenging to engineer because it exhibits complex gradients of structure, composition, biologics, and cellular phenotypes. As a step toward engineering these transitional zones, we initially analyzed how different (topographical or biological) cues affect tenogenic differentiation of adipose-derived stem cells (ADSCs). We immobilized platelet-derived growth factor - BB (PDGF-BB) using polydopamine (PD) chemistry on random and aligned nanofibers and investigated ADSC proliferation and tenogenic differentiation. Immobilized PDGF greatly enhanced the proliferation and tenogenic differentiation of ADSCs; however, nanofiber alignment had no effect. Interestingly, the PDGF immobilized aligned nanofiber group showed a synergistic effect with maximum expression of tenogenic markers for 14 days. We also generated a nanofiber surface with spatially controlled presentation of immobilized PDGF on an aligned architecture, mimicking native tendon tissue. A gradient of immobilized PDGF was able to control the phenotypic differentiation of ADSCs into tenocytes in a spatially controlled manner, as confirmed by analysis of the expression of tenogenic markers and immunofluorescence staining. We further explored the gradient formation strategy by generation of a symmetrical gradient on the nanofiber surface for the generation of a structure mimicking bone-patellar-tendon-bone with provision for gradient immobilization of PDGF and controlled mineralization. Our study reveals that, together with biochemical cues, favorable topographical cues are important for tenogenic differentiation of ADSCs, and gradient presentation of PDGF can be used as a tool for engineering stem cell–based bone-tendon interface tissues.

Freeze-cast Porous Chitosan Conduit for Peripheral Nerve Repair.

A novel freeze-cast porous chitosan conduit for peripheral nerve repair with highly-aligned, double layered porosity, which provides the ideal mechanical and chemical properties was designed, manufactured, and assessed in vivo. Efficacies of the conduit and the control inverted nerve autograft were evaluated in bridging 10-mm Lewis rat sciatic nerve gap at 12 weeks post-implantation. Biocompatibility and regenerative efficacy of the porous chitosan conduit were evaluated through the histomorphometric analysis of longitudinal and transverse sections. The porous chitosan conduit was found to have promising regenerative characteristics, promoting the desired neovascularization, and axonal ingrowth and alignment through a combination of structural, mechanical and chemical cues.

A two-path model of auditory modulation detection using temporal fine structure and envelope cues.

A model using temporal-envelope cues was previously developed to explain perceptual interference effects between amplitude modulation and frequency modulation (FM). As that model could not accurately predict FM sensitivity and the interference effects, temporal fine structure (TFS) cues were added to the model. Thus, following the initial stage of the model consisting of a linear filter bank simulating cochlear filtering, processing was split into an 'envelope path' based on envelope power cues and a 'TFS path' based on a measure of the distribution of time intervals between successive zero-crossings. This yielded independent detectability indices for envelope and TFS cues, which were optimally combined to produce a single decision statistic. Independent internal noises in the envelope and TFS paths were adjusted to match the data. Simulations indicate that TFS cues are required to account for FM data for young normal-hearing listeners and that TFS processing is impaired for both older normal-hearing and hearing-impaired listeners. The role of TFS was further assessed by relating the monaural FM sensitivity to measures of interaural phase difference, commonly assumed to rely on binaural TFS sensitivity. The model demonstrates that binaural TFS sensitivity is considerably lower than monaural TFS sensitivity. Similar to FM thresholds, interaural phase difference sensitivity declined with age and hearing loss, although higher degradations were observed in binaural temporal processing compared to monaural processing. Overall, this model provides a novel tool to explore the mechanisms involved in FM processing in the normal auditory system and the degradations in FM sensitivity with ageing and hearing loss.

In Silico Structural Studies of a Tripeptide Building Block

Molecular self-assembly of peptide building blocks into nanostructures provides minimalistic avenues for developing biologically inspired, “smart” materials with numerous applications in biomedical sciences, photonics, and nanotechnology. Structural investigations of peptide building blocks with a wide range of chemical functionality also hold much promise to decipher their molecular evolution toward the chemical origin of living systems. Our work delves into the structure design and dynamics of a unique tri-peptide unit, which contains three amino acids such as phenylalanine, tyrosine, lysine, and promotes the formation of nanostructures via offsetting the effects of its hydrophobic and hydrophilic components. The results of our computer simulation studies reveal structural cues, demonstrating preferential intra-residue interactions for the favored conformations that could facilitate the self-assembling process. In addition, open-source software and computer simulation methodologies offer attractive opportunities to foster the connection between biophysics and nanotechnology applications and to pursue this interdisciplinary research theme in the liberal arts college setting.

Role of short-time acoustic temporal fine structure cues in sentence recognition for normal-hearing listeners.

Short-time processing was employed to manipulate the amplitude, bandwidth, and temporal fine structure (TFS) in sentences. Fifty-two native-English-speaking, normal-hearing listeners participated in four sentence-recognition experiments. Results showed that recovered envelope (E) played an important role in speech recognition when the bandwidth was > 1 equivalent rectangular bandwidth. Removing TFS drastically reduced sentence recognition. Preserving TFS greatly improved sentence recognition when amplitude information was available at a rate ≥ 10 Hz (i.e., time segment ≤ 100 ms). Therefore, the short-time TFS facilitates speech perception together with the recovered E and works with the coarse amplitude cues to provide useful information for speech recognition.

Structure and assembly cues for rhizospheric nirK- and nirS-type denitrifier communities in long-term fertilized soils

The stimulatory effect of plants on soil denitrification activity has been widely reported, and root-derived carbon (C) is considered a factor contributing to enhanced denitrification activity in the rhizosphere. However, the mechanisms through which root-derived C shapes the rhizospheric denitrifying community structure remains unclear, especially under different soil fertility levels. Here, DNA-based stable isotope probing (DNA-SIP) and Illumina MiSeq sequencing were employed to characterize root-associated denitrifier communities containing nitrite reductase genes (nirS and nirK) in the rhizosphere of wheat grown in soils with three distinct long-term (32-year) fertilization regimes. Fertilization showed a significant impact on the composition of denitrifying communities that actively utilized photosynthetically fixed C in the wheat rhizosphere. In soils amended with inorganic fertilizers, wheat root-derived 13C was mainly assimilated by nirS-type denitrifiers affiliated to Alcaligenaceae and nirK-type denitrifiers affiliated to Phyllobacteriaceae. In contrast, organic fertilization resulted in larger diversity of 13C-labeled denitrifier communities where nirS-type denitrifiers such as Rhodobacteraceae and unclassified Burkholderiales and some unknown nirK-type denitrifiers were more abundant. The nirS-type denitrifier community was found to be more sensitive to the rhizosphere effect than the nirK-type community. Approximately 31% of the 13C-labeled nirS-type denitrifiers were more abundant in the rhizosphere than in the bulk soil, but only 2% of the 13C-labeled nirK-type denitrifiers showed increased abundance in the rhizosphere. The results of this study present direct evidence that root exudates can act as inducible C sources for heterotrophic denitrifying bacteria, but this induction pattern differs between nirS- and nirK-type communities and is dependent on soil fertility level.

Biomimetic Architectures for Peripheral Nerve Repair: A Review of Biofabrication Strategies.

Biofabrication techniques have endeavored to improve the regeneration of the peripheral nervous system (PNS), but nothing has surpassed the performance of current clinical practices. However, these current approaches have intrinsic limitations that compromise patient care. The "gold standard" autograft provides the best outcomes but requires suitable donor material, while implantable hollow nerve guide conduits (NGCs) can only repair small nerve defects. This review places emphasis on approaches that create structural cues within a hollow NGC lumen in order to match or exceed the regenerative performance of the autograft. An overview of the PNS and nerve regeneration is provided. This is followed by an assessment of reported devices, divided into three major categories: isotropic hydrogel fillers, acting as unstructured interluminal support for regenerating nerves; fibrous interluminal fillers, presenting neurites with topographical guidance within the lumen; and patterned interluminal scaffolds, providing 3D support for nerve growth via structures that mimic native PNS tissue. Also presented is a critical framework to evaluate the impact of reported outcomes. While a universal and versatile nerve repair strategy remains elusive, outlined here is a roadmap of past, present, and emerging fabrication techniques to inform and motivate new developments in the field of peripheral nerve regeneration.

Design, Manufacture, and In vivo Testing of a Tissue Scaffold for Permanent Female Sterilization by Tubal Occlusion.

Current FDA-approved permanent female sterilization procedures are invasive and/or require the implantation of non-biodegradable materials. These techniques pose risks and complications, such as device migration, fracture, and tubal perforation. We propose a safe, non-invasive biodegradable tissue scaffold to effectively occlude the Fallopian tubes within 30 days of implantation. Specifically, the Fallopian tubes are mechanically de-epithelialized, and a tissue scaffold is placed into each tube. It is anticipated that this procedure can be performed in less than 30 minutes by an experienced obstetrics and gynaecology practitioner. Advantages of this method include the use of a fully bio-resorbable polymer, low costs, lower risks, and the lack of general anaesthesia. The scaffold devices are freeze-cast allowing for the custom-design of structural, mechanical, and chemical cues through material composition, processing parameters, and functionalization. The performance of the biomaterial and de-epithelialization procedure was tested in an in vivo rat uterine horn model. The scaffold response and tissue-biomaterial interactions were characterized microscopically post-implantation. Overall, the study resulted in the successful fabrication of resilient, easy-to-handle devices with an anisotropic scaffold architecture that encouraged rapid bio-integration through notable angiogenesis, cell infiltration, and native collagen deposition. Successful tubal occlusion was demonstrated at 30 days, revealing the great promise of a sterilization biomaterial.

Integrating soft and hard tissues via interface tissue engineering.

The enthesis, or interface between bone and soft tissues such as ligament and tendon, is prone to injury and often does not heal, even post surgical intervention. Interface tissue engineering represents an integrative strategy for regenerating the native enthesis by functionally connecting soft and hard tissues and thereby improving clinical outcome. This review focuses on integrative and cell-instructive scaffold designs that target the healing of the two most commonly injured soft tissue-bone junctions: tendon-bone interface (e.g., rotator cuff) and ligament-bone interface (e.g., anterior cruciate ligament). The inherent connectivity between soft and hard tissues is instrumental for musculoskeletal motion and is therefore a key design criterion for soft tissue regeneration. To this end, scaffold design for soft tissue regeneration have progressed from single tissue systems to the emerging focus on pre-integrated and functional composite tissue units. Specifically, a multifaceted, bioinspired approach has been pursued wherein scaffolds are tailored to stimulate relevant cell responses using spatially patterned structural and chemical cues, growth factors, and/or mechanical stimulation. Moreover, current efforts to elucidate the essential scaffold design criteria via strategic biomimicry are emphasized as these will reduce complexity in composite tissue regeneration and ease the related burden for clinical translation. These innovative studies underscore the clinical relevance of engineering connective tissue integration and have broader impact in the formation of complex tissues and total joint regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1069-1077, 2018.

Structuring a multi-nodal neural network in vitro within a novel design microfluidic chip.

Neural network formation is a complex process involving axon outgrowth and guidance. Axon guidance is facilitated by structural and molecular cues from the surrounding microenvironment. Micro-fabrication techniques can be employed to produce microfluidic chips with a highly controlled microenvironment for neural cells enabling longitudinal studies of complex processes associated with network formation. In this work, we demonstrate a novel open microfluidic chip design that encompasses a freely variable number of nodes interconnected by axon-permissible tunnels, enabling structuring of multi-nodal neural networks in vitro. The chip employs a partially open design to allow high level of control and reproducibility of cell seeding, while reducing shear stress on the cells. We show that by culturing dorsal root ganglion cells (DRGs) in our microfluidic chip, we were able to structure a neural network in vitro. These neurons were compartmentalized within six nodes interconnected through axon growth tunnels. Furthermore, we demonstrate the additional benefit of open top design by establishing a 3D neural culture in matrigel and a neuronal aggregate 3D culture within the chips. In conclusion, our results demonstrate a novel microfluidic chip design applicable to structuring complex neural networks in vitro, thus providing a versatile, highly relevant platform for the study of neural network dynamics applicable to developmental and regenerative neuroscience.

Bioengineering of the Human Neural Stem Cell Niche: A Regulatory Environment for Cell Fate and Potential Target for Neurotoxicity.

Human neural stem/progenitor cells of the developing and adult organisms are surrounded by the microenvironment, so-called neurogenic niche. The developmental processes of stem cells, such as survival, proliferation, differentiation, and fate decisions, are controlled by the mutual interactions between cells and the niche components. Such interactions are tissue specific and determined by the biochemical and biophysical properties of the niche constituencies and the presence of other cell types. This dynamic approach of the stem cell niche, when translated into in vitro settings, requires building up "biomimetic" microenvironments resembling natural conditions, where the stem/progenitor cell is provided with diverse extracellular signals exerted by soluble and structural cues, mimicking those found in vivo. The neural stem cell niche is characterized by a unique composition of soluble components including neurotransmitters and trophic factors as well as insoluble extracellular matrix proteins and proteoglycans. Biotechnological innovations provide tools such as a new generation of tunable biomaterials capable of releasing specific signals in a spatially and temporally controlled manner, thus creating in vitro nature-like conditions and, when combined with stem cell-derived tissue specific progenitors, producing differentiated neuronal tissue structures. In addition, substantial progress has been made on the protocols to obtain stem cell-derived cell aggregates such as neurospheres and self-assembled organoids.In this chapter, we have assessed the application of bioengineered human neural stem cell microenvironments to produce in vitro models of different levels of biological complexity for the efficient control of stem cell fate. Examples of biomaterial-supported two-dimensional and three-dimensional (2D and 3D) complex culture systems that provide artificial neural stem cell niches are discussed in the context of their application for basic research and neurotoxicity testing.

Structural and functional failure of fibrillin‑1 in human diseases (Review).

Fibrillins (FBNs) are key relay molecules that form the backbone of microfibrils in elastic and non‑elastic tissues. Interacting with other components of the extracellular matrix(ECM), these ubiquitous glycoproteins exert pivotal roles in tissue development, homeostasis and repair. In addition to mechanical support, FBN networks also exhibit regulatory activities on growth factor signalling, ECM formation, cell behaviour and the immune response. Consequently, mutations affecting the structure, assembly and stability of FBN microfibrils have been associated with impaired biomechanical tissue properties, altered cell‑matrix interactions, uncontrolled growth factor or cytokine activation, and the development of fibrillinopathies and associated severe complications in multiple organs. Beyond a panoramic overview of structural cues of the FBN network, the present review will also describe the pathological implications of FBN disorders in the development of inflammatory and fibrotic conditions.