Formation Of Fiber Bundles Research Articles

Effect of Peptide-Polymer Host-Guest Electrostatic Interactions on Self-Assembling Peptide Hydrogels Structural and Mechanical Properties and Polymer Diffusivity.

Peptide-based supramolecular hydrogels are an attractive class of soft materials for biomedical applications when biocompatibility is a key requirement as they exploit the physical self-assembly of short self-assembling peptides avoiding the need for chemical cross-linking. Based on the knowledge developed through our previous work, we designed two novel peptides, E(FKFE)2 and K(FEFK)2, that form transparent hydrogels at pH 7. We characterized the phase behavior of these peptides and showed the clear link that exists between the charge carried by the peptides and the physical state of the samples. We subsequently demonstrate the cytocompatibility of the hydrogel and its suitability for 3D cell culture using 3T3 fibroblasts and human mesenchymal stem cells. We then loaded the hydrogels with two polymers, poly-l-lysine and dextran. When polymer and peptide fibers carry opposite charges, the size of the elemental fibril formed decreases, while the overall level of fiber aggregation and fiber bundle formation increases. This overall network topology change, and increase in cross-link stability and density, leads to an overall increase in the hydrogel mechanical properties and stability, i.e., resistance to swelling when placed in excess media. Finally, we investigate the diffusion of the polymers out of the hydrogels and show how electrostatic interactions can be used to control the release of large molecules. The work clearly shows how polymers can be used to tailor the properties of peptide hydrogels through guided intermolecular interactions and demonstrates the potential of these new soft hydrogels for use in the biomedical field in particular for delivery or large molecular payloads and cells as well as scaffolds for 3D cell culture.

Collective Organization Behaviors of Multi‐Cell Systems Induced by Engineered ECM‐Cell Mechanical Coupling

AbstractCells in vivo are surrounded by fibrous extracellular matrix (ECM), which can mediate the propagation of active cellular forces through stressed fiber bundles and regulate various biological processes. However, the mechanisms for multi‐cellular organization and collective dynamics induced by cell‐ECM mechanical couplings, which are crucial for the development of novel ECM‐based biomaterial for cell manipulation and biomechanical applications, remain poorly understood. Herein, the authors design an in vitro quasi‐3D experimental system and demonstrate a transition between spreading and aggregating in collective organizational behaviors of discrete multi‐cellular systems, induced by engineered ECM‐cell mechanical coupling, with the observed phenomena and underlying mechanisms differing fundamentally from those of cell monolayers. During the process of collective cell organization, the collagen substrate undergoes reconstruction into a dense fiber network structure, which is correlated with local cellular density and consistent with observed enhanced cells' motility; and the weakening of fiber bundle formation within the hydrogel reduces cells’ movement. Moreover, cells can respond to the curvature and shape of the original cell population and form different aggregation patterns. These results elucidate important physical factors involved in collective cell organization and provide important references for potential applications of biomaterials in new therapies and tissue engineering.

Design and fabrication of fiber mesh actuators

Soft actuator performance can be tuned by chemistry or mechanical manipulation, but this adjustability is limited especially in view of their growing technological relevance. Inspired from textile engineering, we designed and fabricated fiber mesh actuators and introduced new features like anisotropic behavior and soft-tissue like elastic deformability. Design criteria for the meshes are the formation of fiber bundles, the angle between fiber bundles in different stacked layers and covalent crosslinks forming within and between fibers at their interfacial contact areas. Through crosslinking the interfiber bond strength increased from a bond transmitting neither axial nor rotational loads (pin joint) to a bond strength capable of both (welded joint). For non-linear elastic stiffening, stacked fiber bundles with four embracing fibers were created forming microstructural rhombus shapes. Loading the rhombus diagonally allowed generation of “soft tissue”-like mechanics. By adjustment of stacking angles, the point of strong increase in stress is tuned. While the highest stresses are observed in aligned and crosslinked fiber mats along the direction of the fiber, the strongest shape-memory actuation behavior is found in randomly oriented fiber mats. Fiber mesh actuators controlled by temperature are of high significance as soft robot skins and as for active patches supporting tissue regeneration.

Glimpsing growing nanotubes

Nanomaterials One-dimensional metal-organic frameworks, also called metal–organic nanotubes (MONTs), are similar to carbon nanotubes but have a wider design space of size and chemical functionality. However, there have been few examples of MONTs, in part because little is known about their growth mechanism. Vailonis et al. synthesized a ligand, 1,4-bis((4H-1,2,4-triazol-4-yl)methyl)naphthalene, that formed a MONT after 5 days of reaction with copper(II) bromide. They also followed this process with liquid-cell transmission electron microscopy and could follow the formation of fiber bundles after initial nucleation. Fitting of this anisotropic growth process to a Lifshitz-Slyozov-Wagner model was consistent with the growth being under thermodynamic control and occurring through a surface-specific monomer-monomer attachment process. Further structural analysis of the material showed that the cell material was the same as the bulk. J. Am. Chem. Soc. 141 , 10177 (2019).

Alignment of Multiple Electrospun Piezoelectric Fiber Bundles Across Serrated Gaps at an Incline: A Method to Generate Textile Strain Sensors

In this paper, we report a new type of electrospinning collector that allows simultaneous collection and alignment of multiple poly(vinylidene fluoride-trifluoroethylene) piezoelectric fiber bundles with a controlled separation. The key enabling feature is the serrated teeth along the edges across an inclined gap as a part of the conductive collector. As a result, the electrical field across the gap is shaped to direct the electrospun fibers to merge into multiple bundles. The sharp points on the serrated teeth provide favorable charge dissipation points and thus fibers are preferentially formed bridging two closest sharp points across the gap. To investigate the effectiveness of serrated teeth on the formation of multiple fiber bundles, three-dimensional finite element simulations are conducted. The corresponding collectors are implemented to experimentally study the resulting electrospun fibers. Both simulation and experimental results suggest that multiple fiber bundles can be formed under the condition of a low teeth pitch to gap distance ratio. Furthermore, a sharper tooth angle results in a higher preferential formation of fiber bundles. Finally, the total electrospinning time should be less than 60 seconds to maintain favorable electric field profile. We also demonstrate that these piezoelectric fiber bundles can serve as ultra-flexible textile sensors.

Mesoscopic Adaptive Resolution Scheme toward Understanding of Interactions between Sickle Cell Fibers

Understanding of intracellular polymerization of sickle hemoglobin (HbS) and subsequent interaction with the membrane of a red blood cell (RBC) is important to predict the altered morphologies and mechanical properties of sickle RBCs in sickle cell anemia. However, modeling the integrated processes of HbS nucleation, polymerization, HbS fiber interaction, and subsequent distortion of RBCs is challenging as they occur at multispatial scales, ranging from nanometers to micrometers. To make progress toward simulating the integrated processes, we propose a hybrid HbS fiber model, which couples fine-grained and coarse-grained HbS fiber models through a mesoscopic adaptive resolution scheme (MARS). To this end, we apply a microscopic model to capture the dynamic process of polymerization of HbS fibers, while maintaining the mechanical properties of polymerized HbS fibers by the mesoscopic model, thus providing a means of bridging the subcellular and cellular phenomena in sickle cell disease. At the subcellular level, this model can simulate HbS polymerization with preexisting HbS nuclei. At the cellular level, if combined with RBC models, the generated HbS fibers could be applied to study the morphologies and membrane stiffening of sickle RBCs. One important feature of the MARS is that it can be easily employed in other particle-based multiscale simulations where a dynamic coarse-graining and force-blending method is required. As demonstrations, we first apply the hybrid HbS fiber model to simulate the interactions of two growing fibers and find that their final configurations depend on the orientation and interaction distance between two fibers, in good agreement with experimental observations. We also model the formation of fiber bundles and domains so that we explore the mechanism that causes fiber branching.

Toughening in electrospun fibrous scaffolds

Electrospun scaffolds mimic the microstructure of structural collagenous tissues and have been widely used in tissue engineering applications. Both brittle cracking and ductile failure have been observed in scaffolds with similarly random fibrous morphology. Finite element analysis can be used to qualitatively examine the mechanics of these differing failure mechanisms. The finite element modeling demonstrates that the noncontinuum deformation of the network structure results in fiber bundle formation and material toughening. Such toughening is accommodated by varying fiber properties, including allowing large failure strains and progressive damage of the fibers.

The ionic products of bioactive glass particle dissolution enhance periodontal ligament fibroblast osteocalcin expression and enhance early mineralized tissue development

This study resulted in enhanced collagen type 1 and osteocalcin expression in human periodontal ligament fibroblasts (hPDLF) when exposed to bioactive glass conditioned media that subsequently may promote early mineralized tissue development. Commercial Bioglass™ (45S5) and experimental bioactive coating glass (6P53-b), were used to make a glass conditioned media (GCM) for comparison to control medium. ICP-MS analysis showed increased concentrations of Ca(2+), PO(4) (3-), Si(4+), and Na(+), for 45S5 GCM and Mg(2+), K(+), Ca(2+), PO(4)(3-), Si(4+), and Na(+) for 6P53-b GCM (relative to control medium). Differentiating hPDLF cultures exposed to 45S5 and 6P53-b GCM showed enhanced expression of collagen type 1 (Col1α1, Col1α2), osteocalcin, and alkaline phosphatase gene expression. These GCM also enhanced osteocalcin protein expression. After 16 d of culture, 45S5 and 6P53-b GCM treated cells showed regions of deep red Alizarin staining, indicating increased Ca within their respective extracellular matrices (ECM), while control-treated cells did not exhibit these features. SEM analysis showed more developed ECM in GCM treated cultures, indicated by multiple tissue layering and abundant collagen fiber bundle formation, while control treated cells did not exhibit these features. SEM analysis showed polygonal structures suggestive of CaP in 45S5 GCM treated cultures. These results indicate the osteogenic potential of bioactive coating glass in periodontal bone defect filling applications.

Instrument-Assisted Cross-Fiber Massage Accelerates Knee Ligament Healing

Controlled laboratory study. To investigate the effects of instrument-assisted cross-fiber massage (IACFM) on tissue-level healing of knee medial collateral ligament (MCL) injuries. Ligament injuries are common and significant clinical problems for which there are few established interventions. IACFM represents an intervention that may mediate tissue-level healing following ligament injury. Bilateral knee MCL injuries were created in 51 rodents, while 7 rodents were maintained as ligament-intact, control animals. IACFM was commenced 1 week following injury and introduced 3 sessions per week for 1 minute per session. IACFM was introduced unilaterally (IACFM-treated), with the contralateral, injured MCL serving as an internal control (nontreated). Thirty-one injured animals received 9 ACFM treatments, while the remaining 20 injured animals received 30 treatments. Ligament biomechanical properties and morphology were assessed at either 4 or 12 weeks postinjury. IACFM-treated ligaments were 43.1% stronger (P<.05), 39.7% stiffer (P<.01), and could absorb 57.1% more energy before failure (P<.05) than contralateral, injured, nontreated ligaments at 4 weeks postinjury. On histological and scanning electron microscopy assessment, IACFM-treated ligaments appeared to have improved collagen fiber bundle formation and orientation within the scar region than nontreated ligaments. There were minimal differences between IACFM-treated and contralateral, nontreated ligaments at 12 weeks postinjury, although IACFM-treated ligaments were 15.4% stiffer (P<.05). IACFM-accelerated ligament healing, possibly via favorable effects on collagen formation and organization, but had minimal effect on the final outcome of healing. These findings are clinically interesting, as there are few established interventions for ligament injuries, and IACFM is a simple and practical therapy technique. J Orthop Sports Phys Ther 2009;39(7):506-514, Epub 24 February 2009. doi:10.2519/jospt.2009.2997.

Gene expression of growth differentiation factors in the developing periodontium of rat molars.

Growth and differentiation factors (GDF) 5, 6, and 7 are known to play roles in tendon and ligament formation, and are therefore probably involved in the formation of periodontal ligament. In this study, we sought to determine temporal and spatial expression of GDF-5, -6, and -7 mRNA in developing periodontal tissue of rat molars using in situ hybridization. GDF gene expression in the periodontal ligament was first detected in cells associated with the initial process of periodontal ligament fiber bundle formation. Gene signals were also detected in cells located along the alveolar bone and cementum surfaces, the insertion sites of periodontal ligaments, during the course of root formation. GDF expression in these cells were down-regulated after completion of root formation. Our results appeared to suggest the involvement of GDF-5, -6, and -7 in the formation of the dental attachment apparatus.

Hindlimb unloading alters ligament healing.

We investigated the hypothesis that hindlimb unloading inhibits healing in fibrous connective tissue such as ligament. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing, and hindlimb-suspended healing. Ambulatory and suspended animals underwent surgical rupture of their medial collateral ligaments, whereas sham surgeries were performed on control animals. After 3 or 7 wk, mechanical and/or morphological properties were measured in ligament, muscle, and bone. During mechanical testing, most suspended ligaments failed in the scar region, indicating the greatest impairment was to ligament and not to bone-ligament insertion. Ligament testing revealed significant reductions in maximum force, ultimate stress, elastic modulus, and low-load properties in suspended animals. In addition, femoral mineral density, femoral strength, gastrocnemius mass, and tibialis anterior mass were significantly reduced. Microscopy revealed abnormal scar formation and cell distribution in suspended ligaments with extracellular matrix discontinuities and voids between misaligned, but well-formed, collagen fiber bundles. Hence, stress levels from ambulation appear unnecessary for formation of fiber bundles yet required for collagen to form structurally competent continuous fibers. Results support our hypothesis that hindlimb unloading impairs healing of fibrous connective tissue. In addition, this study provides compelling morphological evidence explaining the altered structure-function relationship in load-deprived healing connective tissue.

Fluctuations in Self-Assembled Sickle Hemoglobin Fibers

We discuss microscopic measurements of thermal fluctuations in self-assembled sickle hemoglobin fibers. These permit the measurement of various mechanical moduli that control the rheology of the fiber gel responsible for the pathology of the disease. Differential interference contrast microscopy of isolated fibers undergoing equilibrium thermal bending fluctuations leads to estimates of thermal persistence lengths of between 0.23 and 10.4 mm for the most flexible and stiffest fibers, respectively. We argue that this large range may reflect the formation of fiber bundles. If the most flexible fibers are single fibers, then the stiffest measured object is consistent with a “closed shell” hexagonal bundle of 7 single fibers. We estimate the spectrum of persistence lengths associated with bundles consisting of different numbers of single fibers and compare this with the experimental data. The equivalent Young's modulus for the material is 0.1 GPa, less than for structural proteins but much larger than for ext...

α-Smooth muscle actin is expressed by fibroblastic cells of the ovine anterior cruciate ligament and its free tendon graft during remodeling

Contractile fibroblastic cells expressing the α-smooth muscle actin isoform, so-called myofibroblasts, have been identified to play a possible role during the healing of the medial collateral ligament by means of restoring the tissues in situ strain via extracellular matrix contraction. Recently, these cells have also been identified to be a normal part of the human anterior cruciate ligament. It has been hypothesized that myofibroblasts play a role in the wrinkling of the extracellular matrix. The goal of the present study was to identify myofibroblasts in the intact ovine anterior cruciate ligament and a free autologous tendon graft during remodeling after anterior cruciate ligament reconstruction. In 36 mature merino sheep the anterior cruciate ligament was replaced with an ipsilateral Achilles tendon split graft. Midsubstance tissue samples were immunostained for α-smooth muscle actin at 6, 9, 12, 24, 52, and 104 weeks. Myofibroblasts were identified in the intact ovine anterior cruciate ligament as well as in the Achilles tendon graft prior to implantation. During remodeling the first myofibroblasts were found at six weeks within newly formed fiber bundles. At 24, 52, and 104 weeks myofibroblast distribution and cell density were similar to those of the intact ovine anterior cruciate ligament. These findings indicate that α-smooth muscle actin containing fibroblastic cells are a regular part of the intact as well as the remodeled anterior cruciate ligament. There is evidence that myofibroblasts may be involved in maintaining tissue homeostasis in the mature ligament e.g., by means of crimp formation. The presence of these cells during the early remodeling may further indicate that α-smooth muscle actin containing fibroblastic cells are involved in the earliest stages of fiber bundle formation. The role and function of this special cell type for the anterior cruciate ligament needs to be further clarified.

Micromechanics of isolated sickle cell hemoglobin fibers: bending moduli and persistence lengths

Pathogenesis in sickle cell disease depends on polymerization of deoxyhemoglobin S into rod-like fibers, forming gels that rigidify red cells and obstruct the systemic microvasculature. Fiber structure, polymerization kinetics and equilibria are well characterized and intimately related to pathogenesis. However, data on gel rheology, the immediate cause of obstruction, are limited, and models for structure and rheology are lacking. The basis of gel rheology, micromechanics of individual fibers, has never been examined. Here, we isolate fibers by selective depolymerization of gels produced under photolytic deliganding of CO hemoglobin S. Using differential interference contrast (DIC) microscopy, we measure spontaneous, thermal fluctuations in fiber shape to obtain bending moduli (κ) and persistence lengths (λp). Some fibers being too stiff to decompose shape accurately into Fourier modes, we measure deviations of fiber midpoints from mean positions. Serial deviations, sufficiently separated to be independent, exhibit Gaussian distributions and provide mean-squared fluctuation amplitudes from which κ and λp can be calculated. λp ranges from 0.24 to 13 mm for the most flexible and stiffest fibers, respectively. This large range reflects formation of fiber bundles. If the most flexible are single fibers, then λp =13 mm represents a bundle of seven single fibers. Preliminary data on the bending variations of frozen, hydrated single fibers of HbS obtained by electron microscopy indicate that the value 0.24 mm is consistent with the persistence length of single fibers. Young’s modulus is 0.10 GPa, less than for structural proteins but much larger than for extensible proteins. We consider how these results, used with models for cross-linking, may apply to macroscopic rheology of hemoglobin S gels. This new technique, combining isolation of hemoglobin S fibers and measurement of micromechanical properties based on thermal fluctuations and midpoint deviations, can be used to study fibers of mutants, hemoglobin A/S, and mixtures and hybrids of hemoglobin S.

An Enhanced Mutant of Red Fluorescent Protein DsRed for Double Labeling and Developmental Timer of Neural Fiber Bundle Formation

We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.

Changes in the F-actin cytoskeleton during neurosensory bristle development in Drosophila: the role of singed and forked proteins.

Drosophila neurosensory bristle development provides an excellent model system to study the role of the actin-based cytoskeleton in polarized cell growth. We used confocal fluorescence microscopy of isolated thoracic tissue to characterize changes in F-actin that occurred during macrochaete development in wild type flies and mutants that have aberrant bristle morphology. At the earliest stages in wild type bristle development, cortical patches of F-actin were present, but no bundles were observed. Actin bundles began to form at 31% of pupal development and became more prominent as development progressed. The F-actin patches gradually disappeared and were no longer present by 38% of pupal development. The distribution of F-actin in singed3 mutant macrochaetae was indistinguishable from wild type bristles until 35% of development when the actin bundles began to splay and appear ribbon-like. In forked36a bristles, the mutant phenotype was evident at earlier stages of development than the singed3 mutant. Wild type tissue stained with antibodies against the forked protein demonstrated that the forked protein colocalized with F-actin structures found in early and late stage developing macrochaetae. Antibodies against the singed protein showed it appeared to localize with F-actin structures only at later stages in development. These data suggested that the forked gene product was required for the initiation of fiber bundle formation and the singed gene product was required for the maintenance of fiber bundle morphology during bristle development. Similar analyses of singed3/forked36a double mutants provided additional genetic evidence that the forked gene product was required before the singed gene product. Further, the analyses suggested that at least one additional crosslinking protein was present in these bundles.

Neutrophil motility in extracellular matrix gels: mesh size and adhesion affect speed of migration

Polymorphonuclear leukocyte (PMN) migration through tissue extracellular space is an essential step in the inflammatory response, but little is known about the factors influencing PMN migration through gels of extracellular matrix (ECM). In this study, PMN migration within reconstituted gels containing collagen type I or collagen type I supplemented with laminin, fibronectin, or heparin was measured by quantitative direct visualization, resulting in a random motility coefficient (mum a quantitative index for rate of cell dispersion) for the migrating cell population. The random motility coefficient in unsupplemented collagen (0.4 mg/ml) gels was approximately 9 x 10(-9) cm2/s. Supplementing gels with heparin or fibronectin produced a significant decrease in mu, even at the lowest concentrations studied (1 microgram/ml fibronectin or 0.4 microgram/ml heparin). At least 100 micrograms/ml of laminin, or 20% of the total gel protein, was required to produce a similar decrease in mu. Scanning electron microscopy revealed two different gel morphologies: laminin or fibronectin appeared to coat the 150-nm collagen fibers whereas heparin appeared to induce fiber bundle formation and, therefore, larger interstitial spaces. The decrease in mu observed in heparin-supplemented gels correlated with the increased mesh size of the fiber network, but the difference observed in mu for fibronectin- and laminin-supplemented gels did not correlate with either mesh size or the mechanical properties of the gel, as determined by rheological measurements. However, PMNs adhered to fibronectin-coated surfaces in greater numbers than to collagen- or laminin-coated surfaces, suggesting that changes in cell adhesion to protein fibers can also produce significant changes in cell motility within an ECM gel.

Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo.

The production of relatively high quantities of autoantibodies (autoAb) that react with DNA and other intranuclear antigens is characteristic of individuals with systemic lupus erythematosus and other autoimmune diseases. However, the capacity of these Ab to penetrate cells and induce functional perturbations in vivo is not well appreciated. To address this issue, monoclonal (m) anti-DNA Ab (mAb), derived from MRL-lpr/lpr and (NZB x SWR)F1 mice, were administered to normal mice, and the animals were examined for morphologic and functional abnormalities. A subset of five mAb produced intranuclear immunoglobulin deposits in multiple organs. Intranuclear immunoglobulin deposits were also observed after cross-linking the tissue before direct immunofluorescence and after i.v. injection of F(ab')2 fragments of one anti-DNA Ab. This phenomenon was reproducible and was only associated with this subset of autoAb. Furthermore, intranuclear deposits of anti-DNA Ab within glomeruli were associated with morphologic and functional abnormalities including: hypercellularity, epithelial foot process fusion, new fiber bundle formation within the mesangium suggestive of new collagen synthesis, and proteinuria. These results indicate that a subset of autoAb may penetrate cells in vivo to influence normal cellular and nuclear function and to contribute to functional and pathologic abnormalities in individuals with systemic lupus.

The nerve growth factor-inducible large external (NILE) glycoprotein and neural cell adhesion molecule (N-CAM) have distinct patterns of expression in the developing rat central nervous system

The nerve growth factor-inducible large external (NILE) glycoprotein and the neural cell adhesion molecule (N-CAM) have both been implicated in the process of nerve fiber fasciculation. To evaluate the respective roles of the 2 molecules in fiber tract formation, we used immunohistochemical means to compare their distributions in the developing rat central nervous system. In the spinal cord, hindbrain, forebrain, retina, and cerebellum, N-CAM was present on undifferentiated cells in germinal zones as well as on differentiating cells and in nerve fiber tracts. In contrast, NILE was restricted to the developing fiber tracts in all these areas. No fiber tracts were found that were obviously lacking one or the other of the 2 molecules during the period of tract development. However, in all cases except that of the cerebellar molecular layer, nerve fiber tracts appeared to lose NILE and retain N-CAM after the major phases of tract development were completed. The fact that NILE is restricted to nerve fiber tracts during relatively short but crucial phases of tract development suggests that NILE plays a very specific role in the formation of fiber bundles. The more ubiquitous N-CAM molecule may have a more general role in neural histogenesis.

Development of cholinergic properties in nerve cell cultures in the presence of brain extract

Cells dissociated from cerebral hemispheres of 6-day-old chick embryos were cultured either in standard nutrient medium or in the presence of a brain extract from 8-day-old chick embryo. Morphological observations showed the development of bipolar and multipolar neurons in both culture conditions and acetylcholinesterase activity was found in all neuronal cells. Brain extract stimulated the morphological maturation of neurons, expressed by the formation of fiber bundles, fine structural maturation and development of synapses rich in clear vesicles. Furthermore, acetylcholinesterase and choline acetyltransferase activities were higher in the cultures treated with brain extract. In these cultures, the values of choline acetyltransferase activity reached a peak at 10 days and then decreased. These observations are discussed with particular reference to proliferation, maturation and degeneration of cholinergic neurons.