Filament Scaffold Research Articles

Modelling the rheology of living cell cytoplasm: poroviscoelasticity and fluid-to-solid transition

Eukaryotic cell rheology has important consequences for vital processes such as adhesion, migration, and differentiation. Experiments indicate that cell cytoplasm can exhibit both elastic and viscous characteristics in different regimes, while the transport of fluid (cytosol) through the cross-linked filamentous scaffold (cytoskeleton) is reminiscent of mass transfer by diffusion through a porous medium. To gain insights into this complex rheological behaviour, we construct a computational model for the cell cytoplasm as a poroviscoelastic material formulated on the principles of nonlinear continuum mechanics, where we model the cytoplasm as a porous viscoelastic scaffold with an embedded viscous fluid flowing between the pores to model the cytosol. Baseline simulations (neglecting the viscosity of the cytosol) indicate that the system exhibits seven different regimes across the parameter space spanned by the viscoelastic relaxation timescale of the cytoskeleton and the poroelastic diffusion timescale; these regimes agree qualitatively with experimental measurements. Furthermore, the theoretical model also allows us to elucidate the additional role of pore fluid viscosity, which enters the system as a distinct viscous timescale. We show that increasing this viscous timescale hinders the passage of the pore fluid (reducing the poroelastic diffusion) and makes the cytoplasm rheology increasingly incompressible, shifting the phase boundaries between the regimes.

Machine learning to mechanically assess 2D and 3D biomimetic electrospun scaffolds for tissue engineering applications: Between the predictability and the interpretability

Currently, the use of autografts is the gold standard for the replacement of many damaged biological tissues. However, this practice presents disadvantages that can be mitigated through tissue-engineered implants. The aim of this study is to explore how machine learning can mechanically evaluate 2D and 3D polyvinyl alcohol (PVA) electrospun scaffolds (one twisted filament, 3 twisted filament and 3 twisted/braided filament scaffolds) for their use in different tissue engineering applications. Crosslinked and non-crosslinked scaffolds were fabricated and mechanically characterised, in dry/wet conditions and under longitudinal/transverse loading, using tensile testing. 28 machine learning models (ML) were used to predict the mechanical properties of the scaffolds. 4 exogenous variables (structure, environmental condition, crosslinking and direction of the load) were used to predict 2 endogenous variables (Young’s modulus and ultimate tensile strength). ML models were able to identify 6 structures and testing conditions with comparable Young’s modulus and ultimate tensile strength to ligamentous tissue, skin tissue, oral and nasal tissue, and renal tissue. This novel study proved that Classification and Regression Trees (CART) models were an innovative and easy to interpret tool to identify biomimetic electrospun structures; however, Cubist and Support Vector Machine (SVM) models were the most accurate, with R2 of 0.93 and 0.8, to predict the ultimate tensile strength and Young’s modulus, respectively. This approach can be implemented to optimise the manufacturing process in different applications.

2D and 3D PVA electrospun scaffold evaluation for ligament implant replacement: A mechanical testing, modelling and experimental biomechanics approach

The gold standard to characterise anterior cruciate ligament (ACL) implants is through the evaluation of mechanical properties such as Young's modulus or ultimate tensile stress. Currently, no studies have been performed to relate the in-vivo hyper-elastic behaviour of the ACL with the design of tissue engineered ligaments. The aim of this work is to determine the most comparable 2D/3D polyvinyl alcohol (PVA) electrospun structure to the in-vivo mechanical behaviour of the natural ligament. Biomechanics of 12 young participants were captured while daily and high impact activities were performed. A musculoskeletal knee model and kinematic data were used to estimate the in-vivo ACL length and strain. The in-vivo ACL tensile forces were determined with a non-linear force/strain relationship. 2D scaffolds, 1 twisted filament scaffolds, 3 twisted filaments scaffolds and 3 twisted/braided filaments scaffolds were fabricated using electrospinning and characterised morphologically and mechanically using scanning electron microscopy and tensile testing respectively. Cyclic tensile and shear tests were performed in dry and wet conditions to crosslinked and non-crosslinked samples. The hyper-elastic behaviour of our PVA scaffolds was characterised with the Mooney Rivlin model and a non-linear string-based model, and both models compared with the in-vivo mechanical behaviour of the native ACL. Crosslinked 3 twisted/braided filaments scaffolds faithfully mimicked the morphology and the hyper-elastic behaviour of the natural ACL, showed a good resistance to shear loading and remained undegraded in phosphate-buffer saline solution. This study demonstrated, for the first time, that 3 twisted/braided filaments PVA electrospun scaffolds have an excellent potential for ACL replacements.

PH-Responsive Polymer Implants for the Protection of Native Mammals: Assessment of Material Properties and Poison Incorporation on Performance.

Efforts to mitigate the effects of feral cats through the management of remnant or reintroduced populations of threatened species, are often unsuccessful due to predation by control-averse feral cats, or 'problem individuals'. In order to target these animals, we have developed the Population Protecting Implant (PPI). PPIs are designed to be implanted subcutaneously in a native animal. If the animal is preyed upon, and the implant ingested by a feral cat, release of a toxic payload is triggered in the acidic stomach environment and the problem individual is eliminated. We introduce the first toxic implant incorporating the poison sodium fluoroacetate. Manufactured via fluidised-bed spray coating, toxic implants exhibited uniform reverse enteric coatings and low intra-batch variation. Toxic implants were found to exhibit favourable stability at subcutaneous pH in vitro, and rapidly release their toxic payload in vitro at gastric pH. However, limited stability was demonstrated in rats in vivo (~39-230 d), due to the use of a filament scaffold to enable coating and was likely exacerbated by metachromatic interactions caused by 1080. This work highlights that future development of the PPIs should primarily focus on removal of the filament scaffold, to afford implants with increased in vivo stability.

Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation.

As an important sensor in the innate immune system, NLRP3 detects exogenous pathogenic invasions and endogenous cellular damage and responds by forming the NLRP3 inflammasome, a supramolecular complex that activates caspase-1. The three major components of the NLRP3 inflammasome are NLRP3, which captures the danger signals and recruits downstream molecules; caspase-1, which elicits maturation of the cytokines IL-1β and IL-18 and processing of gasdermin D to mediate cytokine release and pyroptosis; and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which functions as a bridge connecting NLRP3 and caspase-1. In this article, we review the structural information that has been obtained on the NLRP3 inflammasome and its components or subcomplexes, with special focus on the inactive NLRP3 cage, the active NLRP3-NEK7 (NIMA-related kinase 7)-ASC inflammasome disk, and the PYD-PYD and CARD-CARD homotypic filamentous scaffolds of the inflammasome. We further implicate structure-derived mechanisms for the assembly and activation of the NLRP3 inflammasome.

In Situ Endothelialization of Free-Form 3D Network of Interconnected Tubular Channels via Interfacial Coacervation by Aqueous-in-Aqueous Embedded Bioprinting.

The challenge of bioprinting vascularized tissues is structure retention and in situ endothelialization. The issue is addressed by adopting an aqueous-in-aqueous 3D embedded bioprinting strategy, in which the interfacial coacervation of the cyto-mimic aqueous two-phase systems (ATPS) are employed for maintaining the suspending liquid architectures, and serving as filamentous scaffolds for cell attachment and growth. By incorporating endothelial cells in the ink phase of ATPS, tubular lumens enclosed by coacervated complexes of polylysine (PLL) and oxidized bacteria celluloses (oxBC) can be cellularized with a confluent endothelial layer, without any help of adhesive peptides. By applying PLL/oxBC ATPS for embedded bioprinting, free-form 3D vascular networks with in situ endothelialization of interconnected tubular lumens are achieved. This simple approach is a one-step process without any sacrificed templates and post-treatments. The resultant functional vessel networks with arbitrary complexity are suspended in liquid medium and can be conveniently handled, opening new routes for the in vitro production of thick vascularized tissues for pathological research, regeneration therapy and animal-free drug development.

Assembling a multi-component and multifunctional integrated filament scaffold based on triaxial 3D bioprinting technology

Currently, most scaffolds produced by 3D printing is based on a single material which limits their wide application in the medical field. In this study, a multi-component and multifunctional shell-core filament 3D scaffold was designed and subsequently produced by triaxial bioprinting technology using sodium alginate, gelatin, collagen, chitosan and platelet-rich plasma (PRP) as biological raw materials. The resulting filamentous 3D scaffold exhibited excellent water absorption and retention capacity, good biocompatibility as well as antibacterial effects. In addition, compared with clinically-used PRP-gel, the filamentous 3D stent could effectively reduce the frequency of PRP administration by 50–75% during wound treatment while also showing better therapeutic effects.

Semiautomated quantification of the fibrous tissue response to complex three-dimensional filamentous scaffolds using digital image analysis.

Fibrosis represents a relevant response to the implantation of biomaterials, which occurs not only at the tissue-material interface (fibrotic encapsulation) but also within the void fraction of complex three-dimensional (3D) biomaterial constructions (fibrotic ingrowth). Usual evaluation of the biocompatibility mostly depicts fibrosis at the interface of the biomaterial using semiquantitative scores. Here, the relations between encapsulation and infiltrating fibrotic growth are poorly represented. Virtual pathology and digital image analysis provide new strategies to assess fibrosis in a more differentiated way. In this study, we adopted a method previously used to quantify fibrosis in visceral organs to the quantification of fibrosis to 3D biomaterials. In a proof-of-concept study, we transferred the "Collagen Proportionate Area" (CPA) analysis from hepatology to the field of biomaterials. As one task of an experimental animal study, we used CPA analysis to quantify the fibrotic ingrowth into a filamentous scaffold after subcutaneous implantation. We were able to demonstrate that the application of the CPA analysis is well suited as an additional fibrosis evaluation strategy for new biomaterial constructions. The CPA method can contribute to a better understanding of the fibrotic interactions between 3D scaffolds and the host tissue responses.

Heterogeneous responses to low level death receptor activation are explained by random molecular assembly of the Caspase-8 activation platform.

Ligand binding to death receptors activates apoptosis in cancer cells. Stimulation of death receptors results in the formation of intracellular multiprotein platforms that either activate the apoptotic initiator Caspase-8 to trigger cell death, or signal through kinases to initiate inflammatory and cell survival signalling. Two of these platforms, the Death-Inducing Signalling Complex (DISC) and the RIPoptosome, also initiate necroptosis by building filamentous scaffolds that lead to the activation of mixed lineage kinase domain-like pseudokinase. To explain cell decision making downstream of death receptor activation, we developed a semi-stochastic model of DISC/RIPoptosome formation. The model is a hybrid of a direct Gillespie stochastic simulation algorithm for slow assembly of the RIPoptosome and a deterministic model of downstream caspase activation. The model explains how alterations in the level of death receptor-ligand complexes, their clustering properties and intrinsic molecular fluctuations in RIPoptosome assembly drive heterogeneous dynamics of Caspase-8 activation. The model highlights how kinetic proofreading leads to heterogeneous cell responses and results in fractional cell killing at low levels of receptor stimulation. It reveals that the noise in Caspase-8 activation—exclusively caused by the stochastic molecular assembly of the DISC/RIPoptosome platform—has a key function in extrinsic apoptotic stimuli recognition.

Fast and reliable advanced two-step pore-size analysis of biomimetic 3D extracellular matrix scaffolds

The tissue microenvironment is a major contributor to cellular functions, such as cell adhesion, migration and invasion. A critical physical parameter for determining the effect of the microenvironment on cellular functions is the average pore-size of filamentous scaffolds, such as 3D collagen fiber matrices, which are assembled by the polymerization of biopolymers. The scaffolds of these matrices can be analyzed easily by using state-of-the-art laser scanning confocal imaging. However, the generation of a quantitative estimate of the pore-size in a 3D microenvironment is not trivial. In this study, we present a reliable and fast analytical method, which relies on a two-step 3D pore-size analysis utilizing several state-of-the-art image analysis methods, such as total variation (TV) denoising and adaptive local thresholds, and another crucial parameter, such as pore-coverage. We propose an iterative approach of pore-size analysis to determine even the smallest and obscure pores in a collagen scaffold. Additionally, we propose a novel parameter, the pseudo-pore-size, which describes a virtual scaffold porosity. In order to validate the advanced two-step pore-size analysis different types of artificial collagens, such as a rat and bovine mixture with two different collagen concentrations have been utilized. Additionally, we compare a traditional approach with our method using an artificially generated network with predefined pore-size distributions. Indeed, our analytical method provides a precise, fast and parameter-free, user-independent and automatic analysis of 3D pore topology, such as pore-sizes and pore-coverage. Additionally, we are able to determine non-physiological network topologies by taking the pore-coverage as a goodness-of-fit parameter.

Assembly mechanism of the CARMA1–BCL10–MALT1–TRAF6 signalosome

The CARMA1-BCL10-MALT1 (CBM) signalosome is a central mediator of T cell receptor and B cell receptor-induced NF-κB signaling that regulates multiple lymphocyte functions. While caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1) nucleates B cell lymphoma 10 (BCL10) filament formation through interactions between CARDs, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a paracaspase with structural similarity to caspases, which recruits TNF receptor-associated factor 6 (TRAF6) for K63-linked polyubiquitination. Here we present cryo-electron microscopy (cryo-EM) structure of the BCL10 CARD filament at 4.0-Å resolution. The structure redefines CARD-CARD interactions compared with the previous EM structure determined from a negatively stained sample. Surprisingly, time-lapse confocal imaging shows that BCL10 polymerizes in a unidirectional manner. CARMA1, the BCL10 nucleator, serves as a hub for formation of star-shaped filamentous networks of BCL10 and significantly decreases the lag period of BCL10 polymerization. Cooperative MALT1 interaction with BCL10 filaments observed under EM suggests immediate dimerization of MALT1 in the BCL10 filamentous scaffold. In addition, TRAF6 cooperatively decorates CBM filaments to form higher-order assemblies, likely resulting in all-or-none activation of the downstream pathway. Collectively, these data reveal biophysical mechanisms in the assembly of the CARMA1-BCL10-MALT1-TRAF6 complex for signal transduction.

Human Adipose Stem Cells Differentiated on Braided Polylactide Scaffolds Is a Potential Approach for Tendon Tissue Engineering.

Growing number of musculoskeletal defects increases the demand for engineered tendon. Our aim was to find an efficient strategy to produce tendon-like matrix in vitro. To allow efficient differentiation of human adipose stem cells (hASCs) toward tendon tissue, we tested different medium compositions, biomaterials, and scaffold structures in preliminary tests. This is the first study to report that medium supplementation with 50 ng/mL of growth and differentiation factor-5 (GDF-5) and 280 μM l-ascorbic acid are essential for tenogenic differentiation of hASCs. Tenogenic medium (TM) was shown to significantly enhance tendon-like matrix production of hASCs compared to other tested media groups. Cell adhesion, proliferation, and tenogenic differentiation of hASCs were supported on braided poly(l/d)lactide (PLA) 96l/4d copolymer filament scaffolds in TM condition compared to foamed poly(l-lactide-co-ɛ-caprolactone) (PLCL) 70L/30CL scaffolds. A uniform cell layer formed on braided PLA 96/4 scaffolds when hASCs were cultured in TM compared to maintenance medium (MM) condition after 14 days of culture. Furthermore, total collagen content and gene expression of tenogenic marker genes were significantly higher in TM condition after 2 weeks of culture. The elastic modulus of PLA 96/4 scaffold was more similar to the elastic modulus reported for native Achilles tendon. Our study showed that the optimized TM is needed for efficient and rapid in vitro tenogenic extracellular matrix production of hASCs. PLA 96/4 scaffolds together with TM significantly stimulated hASCs, thus demonstrating the potential clinical relevance of this novel and emerging approach to tendon injury treatments in the future.

Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.

Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis.

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1-RIP1 interaction is dispensable for necroptosis; RIP1-RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1-RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3-RIP3 interaction is required for necroptosis and RIP3-RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1-RIP3 heterodimer itself cannot induce necroptosis, the RIP1-RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1-RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.

The titin A-band rod domain is dispensable for initial thick filament assembly in zebrafish

The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely accepted hypotheses for this process include the “molecular ruler” model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the “premyofibril” model, which proposes that thick filament formation does not require titin, but that a “premyofibril” consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the “molecular ruler” model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy.

Redundancy and Cooperativity in the Mechanics of Compositely Crosslinked Cytoskeletal Networks

The actin cytoskeleton contains many types of crosslinkers---for example, crosslinkers that allow for free rotations between filaments and others that constrain the angle between crossing filaments. The mechanical interplay between different crosslinkers is an open issue in cytoskeletal mechanics. We develop a theoretical framework to study such a compositely crosslinked actin network to address this issue. We use an effective medium theory and computational modeling to carry out our investigations, with very good agreement between the two.We find that the addition of angle-constraining crosslinkers, acting cooperatively with freely-rotating crosslinkers, allows the network to attain mechanical rigidity (a nonzero shear modulus) as soon as there exists a connected percolating cluster. This finding suggests the optimal conditions for the actin cytoskeleton to become rigid and be able to bear and transmit forces with the minimal amount of material possible. Without angle-constraining crosslinkers, the rigidity percolation threshold requires a higher density of filaments such that the transmission of forces is not as efficient. We further demonstrate that the introduction of angle-constraining crosslinks in flexible filament networks results in mechanical response similar to just freely-rotating crosslinked semiflexible filaments indicating redundancy. Our results also impact upon tissue engineering by providing design principles for developing filamentous scaffolds with tunable mechanical properties.

Bacillus subtilis MreB Orthologs Self-Organize into Filamentous Structures underneath the Cell Membrane in a Heterologous Cell System

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

Nuclear Matrix Proteome Analysis of Drosophila melanogaster

The nucleus is a highly structured organelle and contains many functional compartments. Although the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat, although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we found structural proteins, chaperones, DNA/RNA-binding proteins, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. Comparison of the two-dimensional profile of NuMat proteome from different developmental stages of Drosophila embryos showed that less than half of the NuMat proteome is constant, and the rest of the proteins are stage-specific dynamic components. These NuMat dynamics suggest a possible functional link between NuMat and embryonic development. Finally, we also showed that a subset of NuMat proteins remains associated with the mitotic chromosomes, implicating their role in mitosis and possibly the epigenetic cellular memory. NuMat proteome analysis provides tools and opens up ways to understand nuclear organization and function.

Environmental factors involved in axonal regeneration following spinal cord transection in rats

A recent study of a rat model treated with grafted collagen filament (CF) after spinal cord transection showed dramatic recovery of motor function but did not report on the acute-stage phenomenon. In the present study, we describe molecular and histological aspects of the axonal regeneration process during the acute stage following spinal cord transection. The spinal cord of 8-week-old rats was completely transected, and a scaffold of almost the same size as the resected portion was implanted in the gap. Changes in the mRNA expression of four neurotrophic factors [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, and glial cell-derived neurotrophic factor (GDNF)] were analyzed after 72 h. The expression of BDNF and NT-3 mRNA increased significantly in the CF-grafted group compared to the nongrafted group. Immunostaining for BDNF and NT-3 revealed that cells positive for these neurotrophic factors extended along the collagen filaments in the CF-grafted group. Similarly, astrocytes extended into the collagen filament scaffold together with the neurotrophic factors and partly across a border line. These findings indicate that collagen filament helps to reduce scar tissue, supports the expression of neurotrophic factors, and serves as a scaffold for the outgrowth of regenerating axons.

Enhancement of In Vitro Capillary Tube Formation by Substrate Nanotopography.

Vascular engineering remains a key thrust in advancing the field of tissue engineering of highly vascularized, complex, metabolic organs. A wide variety of strategies have been employed to control the formation of organized vascular structures in vitro and in vivo. Some of these methods include, but are not limited to, controlled growth factor delivery,[1] filamentous scaffold geometry,[2] protein micropatterning,[3] and enhanced scaffold biomaterials.[4] Many of these approaches are motivated by biomimicry of the in vivo microenvironment. Extracellular matrix (ECM) proteins, both in vitro and in vivo, provide mammalian cells with biophysical cues including specific surface chemistry and rich three-dimensional surface topography[5] with features on the nanometer length scale.[6] ECM substrates provide chemical and physical external cues that dictate a variety of cell responses. Therefore, it is not only the milieu of soluble, diffusible factors, but also the adhesive, mechanical interactions with scaffolding materials, both natural and synthetic, that control select cell functions including cell attachment, migration, proliferation, differentiation, and regulation of genes.[7–9] We hypothesized that physical features on nanofabricated substrates could promote the organization of endothelial cell lineages into well-defined vascular structures in vitro by inducing the contact guidance phenomenon, which is known to affect the morphology of endothelial cells.[10–12]We found that endothelial progenitor cells (EPCs) responded to ridge-groove grating of 1200 nm in period and 600 nm in depth through alignment, elongation, reduced proliferation, and enhanced migration. Although endothelial- specific markers were not significantly altered, EPCs cultured on substrate nanotopography formed supercellular band structures after 6 d. Furthermore, an in vitro Matrigel assay led to enhanced capillary tube formation and organization.