PC12 Cell Attachment Research Articles

Development of a Bioactive Scaffold based on NGF Containing PCL/Chitosan Nanofibers for Nerve Regeneration

Nanofibers are used in a wide range of applications, including scaffolds for tissue engineering. Electrospinning is a promising technique to fabricate nanofibrous scaffolds capable of supporting cell attachment and growth. Nanofibers with biomimetic alignment could also guide neural cell growth and orientation of cell processes necessary for peripheral nerve regeneration. This study fabricated aligned nanofibers of polycaprolactone/chitosan (PCL/CS) scaffolds and immobilized nerve growth factor (NGF) on them via dopamine coating mediated bonds to confer bioactivity to the scaffold and support attachment and growth of PC12 cells. The results showed that PCL/CS nanofibrous scaffolds revealed appropriate mechanical and surface properties. Cells remained viable on the scaffolds, and surface-modified aligned nanofibrous scaffolds interacted better with the cells, inducing neural morphology and orientation. Immobilization of NGF via polydopamine (PD) on nanofibers' surface proved to be a proper method to enhance PC12 cell attachment and proliferation. Thus, this construct could potentially be used as a scaffold for peripheral nerve regeneration.

The effect of surface modification of poly-lactide-co-glycolide/carbon nanotube nanofibrous scaffolds by laminin protein on nerve tissue engineering.

The presence of biological cues to promote the attachment, proliferation, and differentiation of neuronal cells is important in the process of nerve regeneration. In this study, laminin as a neurite promoting protein, has been used to modify poly-lactide-co-glycolide/carbon nanotube (PLGA/CNT) electrospun nanofibrous scaffolds by means of either mussel-inspired poly(dopamine) (PD) coating or via direct physical adsorption as a simple route for the functionalization of biomaterials. The laminin-modified scaffolds were characterized by a combination of field emission scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, and contact angle measurements. Subsequently, various properties of scaffolds such as degradation time, amount of attached laminin and the rate of CNT release were investigated. The synergistic effect of topographical and biological cues for PC12 cell attachment, proliferation, and differentiation were then studied by SEM and confocal microscopy. The results of degradation study showed that laminin-modified scaffolds were biodegradable with good structural integrity that persisted about 4 weeks. The amount of laminin attached to the PLGA/CNT and PLGA/CNT-PD scaffolds was 3.12 ± 0.6 and 3.04 ± 071 μg per mg of the scaffold, respectively. Although laminin-modified scaffolds could improve cell proliferation identically, neurite extensions on the PLGA/CNT scaffold modified via PD coating (PLGA/CNT-PD-lam scaffold) were significantly longer than those observed on PLGA/CNT scaffold modified via physical adsorption (PLGA/CNT-lam scaffold) and unmodified scaffolds. Together, these results indicated that surface modification via PD coating could be a promising strategy to fabricate biomimetic scaffolds capable of sustaining longer neuronal growth for nerve tissue engineering.

Physiochemical and morphological dependent growth of NIH/3T3 and PC-12 on polyaniline-chloride/chitosan bionanocomposites

Biomimetic scaffolds inspired by fields and forces of the natural environment of cells is essential in tissue engineering. This study reports on controlled growth of two model cell lines, NIH/3T3 (promiscuous, fibroblast) and PC-12 (electroresponsive, neural progenitor) cells, given electrical and topographical cues that were delivered from a bionanocomposite of polyaniline-chloride and chitosan (PAn-Cl/CHI). The conductivity and morphology of the scaffold were controlled by varying the wt% of PAn-Cl (0–50 wt%) in CHI and processing methods, air-drying (nanofeatured) versus lyophilization (microporous-reticulated), respectively. Bionanocomposites supported the growth of both cell types independent of the availability of receptor-mediated ligands (laminin). NIH/3T3 cells were less elongated on lyophilized (microporous-reticulated) and more conductive (higher wt% PAn-Cl) composites. PC-12 cells had higher viability and less aggregation when grown on conductive substrates. Air-dried bionanocomposites were more supportive of growth but not attachment of PC-12 cells, suggesting that processing of composites could provide an additional level of engineering control to alter the PC-12 cell attachment and growth. In general, PC-12 cells responded more distinctly and dramatically to the substrate properties than NIH/3T3 cells, supporting a clear role for electrical conductivity on neural cell behavior. Nerve growth factor(NGF)-induced differentiation of PC-12 cells resulted in extensive neurite extension in the presence of adsorbed laminin. In a substrate composition-dependent manner, extension and rate of neurite outgrowth were higher when cultured on the conductive substrates. Overall, this study demonstrates the suitability of conductive PAn-Cl/CHI scaffold to host different cell types and support their responses.

A three-layered hollow tubular scaffold as an enhancement of nerve regeneration potential

A significant clinical challenge in the surgery of peripheral nervous system injured via accidents and natural disease is development of biomimetic grafts which could potentially promote nerve repair and regeneration. Although various engineered neural tissue scaffolds have been proposed to support the neural cell functions, they have not been able to instantaneously mimic the whole characteristics of endogenous microenvironment. In this study, we proposed a three-layered tubular scaffold which could provide appropriate electrical, mechanical and biological properties for peripheral nerve engineering. While the inter layer was graphene (Gr) embedded alginate-polyvinyl alcohol (AP-Gr) fibrous scaffold with well-defined anisotropy, the outer layer was double network scaffold of polycaprolactone fumarate (PCLF) and eggshell membrane (ESM). These two layers were attached together using a polycaprolactone (PCL) fibrous membrane, a middle layer, via a simple melting process. Results showed that while the electrical conductivity of the three-layered scaffold was similar to that of AP-Gr fibrous layer, the strength of the three-layered scaffold was significantly improved compared to AP-Gr and ESM-PCLF (1.5 and 1.1 times, respectively) attributed to well attachment of the two layers. As a proof-of-concept, PC12 cell attachment, proliferation, and alignment were studied on the developed three-layered scaffold. The majority of the cells (55%) aligned (<20°) along the major axis of fibers features. Furthermore, electrical stimulation revealed positive effect on the alignment of PC12 cells and change in the cell morphology. With the ease of fabrication and mechanical robustness, the three-layered scaffold of AP-Gr and ESM-PCLF might be utilized as a versatile system for the engineering of peripheral nerve tissue.

Modulation of the mechanical, physical and chemical properties of polyvinylidene fluoride scaffold via non-solvent induced phase separation process for nerve tissue engineering applications

The aim of this research was to develop microporous poly(vinylidene fluoride) (PVDF) scaffolds with an intrinsic electrical property, via the combination of non-solvent induced phase separation (NIPS) and thermal induced phase separation (TIPS) process referred as N-TIPS method. For this purpose, the effects of non-solvent incorporation (distilled water) in the solvent composition (N,N-dimethylformamide (DMF)), immersion time at coagulation bath (1, 3, 6 and 24 h) as well as coagulation bath temperature (−10, 0 and 20 °C) and composition (DMF:water volume ratio = 2:6 and 6:4) on the properties of the produced scaffolds were investigated. Results confirmed that N-TIPS processing parameters had a profound effect on the morphological, mechanical, physical and thermal properties of the PVDF scaffolds. For instance, with increased bath temperature, the formation of three-dimensional bi-continuous scaffold with average pore size of 4.2 ± 0.6 μm was achieved, whereas increase in the immersion time in coagulation bath from 1 to 24 h induced cellular morphology with a larger pore size. The formation of relatively small pore size and uniform foam-like structure at 3 h soaking in coagulation bath showed to improved mechanical properties of the scaffolds. It was also found that toughness of the scaffolds significantly promoted from 27.5 ± 16.4 MPa (after 1 h soaking) to 155.2 ± 25.4 MPa (after 3 h soaking). Moreover, depending on the functional parameters of N-TIPS process, β phase fraction and crystallinity of the PVDF scaffolds were in the range of 61–87% and 30–47%, respectively. Remarkably, 3 h soaking of PVDF polymer solution in coagulation bath with composition of 6:4 (D-3h-64 scaffold) significantly enhanced the crystallinity (47.03%) and β phase fraction (87.9%) and reduced crystallite size of PVDF polymer. The PC12 cell attachment and proliferation on PVDF scaffolds prepared at various parameters were also investigated. Noticeably, D-3h-64 scaffold with enhanced crystallinity and β phase fraction and significantly higher toughness could promote cell spreading and proliferation. The results presented in this study show a great potential of PVDF scaffolds with desired properties for nerve tissue engineering application.

Pheochromocytoma (PC12) Cell Response on Mechanobactericidal Titanium Surfaces.

Titanium is a biocompatible material that is frequently used for making implantable medical devices. Nanoengineering of the surface is the common method for increasing material biocompatibility, and while the nanostructured materials are well-known to represent attractive substrata for eukaryotic cells, very little information has been documented about the interaction between mammalian cells and bactericidal nanostructured surfaces. In this study, we investigated the effect of bactericidal titanium nanostructures on PC12 cell attachment and differentiation—a cell line which has become a widely used in vitro model to study neuronal differentiation. The effects of the nanostructures on the cells were then compared to effects observed when the cells were placed in contact with non-structured titanium. It was found that bactericidal nanostructured surfaces enhanced the attachment of neuron-like cells. In addition, the PC12 cells were able to differentiate on nanostructured surfaces, while the cells on non-structured surfaces were not able to do so. These promising results demonstrate the potential application of bactericidal nanostructured surfaces in biomedical applications such as cochlear and neuronal implants.

Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips

Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter—towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

Preparation, characterisation, and in vitro evaluation of electrically conducting poly(ɛ-caprolactone)-based nanocomposite scaffolds using PC12 cells.

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

A laminin-2-derived peptide promotes early-stage peripheral nerve regeneration in a dual-component artificial nerve graft

The DLTIDDSYWYRI motif (Ln2-P3) of human laminin-2 has been reported to promote PC12 cell attachment through syndecan-1; however, the in vivo effects of Ln2-P3 have not been studied. In Schwann cells differentiated from skin-derived precursors, the peptide was effective in promoting cell attachment and spreading in vitro. To examine the effects of Ln2-P3 in peripheral nerve regeneration in vivo, we developed a dual-component poly(p-dioxanone) (PPD)/poly(lactic-co-glycolic acid) (PLGA) artificial nerve graft. The novel graft was coated with scrambled peptide or Ln2-P3 and used to bridge a 10 mm defect in rat sciatic nerves. The dual-component nerve grafts provided tensile strength comparable to that of a real rat nerve trunk. The Ln2-P3-treated grafts promoted early-stage peripheral nerve regeneration by enhancing the nerve regeneration rate and significantly increased the myelinated fibre density compared with scrambled peptide-treated controls. These findings indicate that Ln2-P3, combined with tissue-engineering scaffolds, has potential biomedical applications in peripheral nerve injury repair.

Real-time monitoring of extracellular matrix-mediated PC12 cell attachment and proliferation using an electronic biosensing device

The attachment and proliferation of a well-established, neuron-like cell line, rat pheochromocytoma (PC12) cells, on different extracellular matrices (ECMs) was monitored using cellular impedance sensing (CIS). Commonly used ECMs, including fibronectin, laminin, poly-L: -lysine, collagen and poly-L: -lysine followed by laminin, in addition to DMEM cell culture media alone as a control, were studied: CIS identified the dynamic progress of the adhesion and proliferation of the cells on different ECMs. Among these modified ECM surfaces, the laminin- and poly-L: -lysine/laminin-modified surfaces were the best suited for the neuron-to-electrode surface attachment and proliferation, which was confirmed by MTT assays and a scanning electron microscopy analysis. This work provides a simple method to study neuron cell/ECM interactions in a real-time, label-free, and quantitative manner.

Porous and Electrically Conductive Polypyrrole−Poly(vinyl alcohol) Composite and Its Applications as a Biomaterial

Bulk modification of polypyrrole (PPY) with poly(vinyl alcohol) (PVA) was carried out by the electropolymerization of pyrrole in the presence of PVA in the reaction solution, with tetraethylammonium perchlorate (TEAP) as the electrolyte. The surface morphology of the as-synthesized PPY-TEAP-PVA film was investigated using scanning electron microscopy, and the film was further characterized using X-ray photoelectron spectroscopy, electrical conductivity, the water contact angle, and BET surface area measurements. The PPY-TEAP-PVA composite is electrically conductive, hydrophilic, and microporous with a high surface area. Its potential as a biomaterial was investigated with respect to its blood compatibility and function as a substrate for biosensor fabrication and cell culture. The presence of PVA in the film attenuates blood protein adsorption, and the porous nature of the PPY-TEAP-PVA film results in a 10-fold increase in the amount of glucose oxidase covalently immobilized on the film over that on a nonporous PPY film. PC12 cell attachment and growth on the PPY-TEAP-PVA film was also shown to be enhanced compared with that on tissue culture polystyrene. The attached cells proliferated and formed a monolayer on the film surface after 48 h of seeding.

Dystrophin Dp71 in PC12 cell adhesion

Previously, we reported that PC12 cells with decreased Dp71 expression (antisense-Dp71 cells) display deficient nerve-growth-factor-induced neurite outgrowth. In this study, we show that disturbed neurite outgrowth of antisense-Dp71 cells is accompanied by decreased adhesion activity on laminin, collagen and fibronectin. In wild-type cells, the immunostaining of Dp71 and beta1-integrin overlaps in the basal area contacting the substrate, but staining of both proteins decrease in the antisense-Dp71 cells. Morphology of antisense-Dp71 cells at the electron microscopic level is characterized by the lack of filopodia, cellular projections involved in adhesion. Our findings suggest that Dp71 is required for the efficient PC12 cell attachment to beta1-integrin-dependent substrata and that decreased adhesion activity of the antisense-Dp71 cells could determine their deficiency to extend neurites.

Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain.

Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering.

Nitric Oxide Mediates Laminin-Induced Neurite Outgrowth in PC12 Cells

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.

Inhibition of PC12 cell attachment and neurite outgrowth by detergent solubilized CNS myelin proteins.

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN-1 directed against the neurite growth inhibiting proteins NI-35 and NI-250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.

Neuronal cell attachment to fluorinated ethylene propylene films with covalently immobilized laminin oligopeptides YIGSR and IKVAV. II.

Material surfaces that can mediate cellular interactions by the coupling of specific cell membrane receptors may allow for the design of a biomaterial that can control cell attachment, differentiation, and tissue organization. Cell adhesion proteins have been shown to contain minimum oligopeptide sequences that are recognized by cell surface receptors and can be covalently immobilized on material surfaces. In this study, cell attachment to fluorinated ethylene propylene (FEP) films functionalized with the laminin-derived oligopeptides, YIGSR and a 19-mer IKVAV-containing sequence, was assessed using NG108-15 neuroblastoma and PC12 cells. A radiofrequency glow discharge (RFGD) process that replaces the FEP surface fluorine atoms with reactive hydroxyl functionalities was used to activate the film surfaces. The oligopeptides were then covalently coupled to the surface by their C-terminus using a standard nucleophilic substitution reaction. The covalent attachment of the oligopeptides to the FEP surface was verified using electron spectroscopy for chemical analysis (ESCA). Receptor-mediated NG108-15 cell attachment on the YIGSR-modified films was determined using competitive binding assays. Average cell attachment on the oligopeptide immobilized films in medium containing soluble CDPGYIGSR was reduced by approximately a factor of 2, compared to cell attachment in serum-free medium alone. No significant decrease in cell attachment was noted in medium containing the mock oligopeptide sequence CDPGYIGSK. FEP films immobilized with the 19-mer IKVAV sequence demonstrated a higher percentage of receptor mediated cell attachment on the film surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial control of neuronal cell attachment and differentiation on covalently patterned laminin oligopeptide substrates

The spatial control of neuronal cell attachment and differentiation via specific receptor mediated interactions, may provide an effective means for the in vitro reconstruction of neuronal cell architecture. In this study, receptor-specific oligopeptide sequences derived from the extracellular matrix (ECM) molecule laminin, a potent neural cell attachment and differentiation promoter were covalently bound on fluorinated ethylene propylene (FEP) films. The degree of receptor-specific cell attachment and the ability to spatially control neurite outgrowth by covalently patterning the oligopeptide sequences on the FEP film surface were assessed.FEP films were first chemically activated with a Radio Frequency Glow Discharge (RFGD) process that covalently replaces the surface fluorine atoms with reactive hydroxyl groups. Oligopeptides containing the YIGSR sequence from the B1 chain of laminin and the water soluble oligopeptide containing the IKVAV sequence (CSRARKQAASIKVAVSADR) from the A chain were covalently bound to the hydroxylated FEP films. Electron Spectroscopy for Chemical Analysis (ESCA) verified the covalent attachment of the oligopeptides to the material surface.The degree of receptor mediated NG108-15 cell attachment on immobilized CDPGYIGSR films was determined using competitive binding media. A 78% reduction in cell attachment was observed on films containing CDPGYIGSR in the cell plating medium. Only a 23% reduction in cell attachment was noted on films plated in medium containing a mock CDPGYIGSK sequence. FEP films immobilized with the IKVAV oligopeptide sequence were shown to mediate PC12 cell attachment and a competitive binding medium also significantly attenuated cell attachment on the immobilized films.The spatial patterning of these oligopeptide sequences to the FEP surface was shown to localize cell attachment and neurite extension on the patterned pathways. The surrounding unmodified FEP surface was inhibitory in serum containing medium and prevented cellular interactions outside the oligopeptide modifications. The spatial immobilization of laminin oligopeptides on FEP films provides a means to organize the attachment and differentiation of neuronal cells in a receptor-specific manner.

Laminin “A” chain fragment of MR 43 kilodalton contains PC12 cell attachment promoting site

In the present study, the ability of a 43 K fragment, originating from the long arm of the 400 K laminin “A” chain (Rao and Kefalides, manuscript in press), to promote the attachment and de novo neurite outgrowth by the pheochromocytoma cell line, (PC12) was studied. Unprimed PC12 cells (not treated with nerve growth factor, NGF), just as the primed cells (treated with NGF),rapidly and efficiently attach to the laminin fragment but less efficiently to the intact laminin coated bacteriological plastic surfaces. The neurite outgrowth was continuous from primed cells attached to both laminin and laminin fragment substrates, when cultured for four days in the presence of NGF. On the other hand, no significant neurite outgrowth was observed from unprimed PC12 cells attached to the laminin fragment coated bacteriological dishes, in the absence of NGF. These data suggest that the 43 K laminin “A” chain fragment contains the PC12 cell attachment promoting site.

Induction of cell attachment and morphological differentiation in a pheochromocytoma cell line and embryonal sensory cells by the extracellular matrix

Embryonic chicken sensory cells from dorsal root ganglia and a clonal line of pheochromocytoma cells (PC-12) extended neuronal-like processes within 24 hr of seeding on a naturally produced, basement membrane-like extracellular matrix (ECM) in the absence of nerve growth factor (NGF). Plating on ECM also induced a rapid cell attachment and flattening of these cells and supported the survival of embryonic sensory cells in primary cultures. Unlike the effect of NGF on PC-12 cells, the ECM-induced morphological differentiation was transient and led to disintegration and degeneration of processes bearing PC-12 cells. The ECM-induced morphological differentiation was not inhibited by anti-NGF antibodies, and the cells retained their ability to bind and internalize NGF in a manner similar to that observed on plastic. PC-12 cell attachment and flattening occurred on dishes coated with collagen type IV in a way similar to that observed on ECM, but precoating the dishes with fibronectin had no effect. Extension of cell processes was not induced by either substrate. Morphological differentiation but not the induction of cell adhesion and flattening was inhibited by either prefixation with glutaraldehyde, oxidation with periodate, or preexposure to concanavalin A of the ECM, suggesting that the ECM and in particular its sugar moieties play an active role in the induction of neurite outgrowth. It is suggested that close contact with the ECM provides chemical or mechanical cues that permit contactmediated elongation and directed growth of both embryonic and regenerating nerve fibers.