Neurite Extension In PC12 Cells Research Articles

A Biomimetic Nonwoven-Reinforced Hydrogel for Spinal Cord Injury Repair.

In clinical trials, new scaffolds for regeneration after spinal cord injury (SCI) should reflect the importance of a mechanically optimised, hydrated environment. Composite scaffolds of nonwovens, self-assembling peptides (SAPs) and hydrogels offer the ability to mimic native spinal cord tissue, promote aligned tissue regeneration and tailor mechanical properties. This work studies the effects of an aligned electrospun nonwoven of P11-8—enriched poly(ε-caprolactone) (PCL) fibres, integrated with a photo-crosslinked hydrogel of glycidylmethacrylated collagen (collagen-GMA), on neurite extension. Mechanical properties of collagen-GMA hydrogel in compression and shear were recorded, along with cell viability. Collagen-GMA hydrogels showed J-shaped stress–strain curves in compression, mimicking native spinal cord tissue. For hydrogels prepared with a 0.8-1.1 wt.% collagen-GMA concentration, strain at break values were 68 ± 1–81 ± 1% (±SE); maximum stress values were 128 ± 9–311 ± 18 kPa (±SE); and maximum force values were 1.0 ± 0.1–2.5 ± 0.1 N (±SE). These values closely mimicked the compression values for feline and porcine tissue in the literature, especially those for 0.8 wt.%. Complex shear modulus values fell in the range 345–2588 Pa, with the lower modulus hydrogels in the range optimal for neural cell survival and growth. Collagen-GMA hydrogel provided an environment for homogenous and three-dimensional cell encapsulation, and high cell viability of 84 ± 2%. Combination of the aligned PCL/P11-8 electrospun nonwoven and collagen-GMA hydrogel retained fibre alignment and pore structure, respectively, and promoted aligned neurite extension of PC12 cells. Thus, it is possible to conclude that scaffolds with mechanical properties that both closely mimic native spinal cord tissue and are optimal for neural cells can be produced, which also promote aligned tissue regeneration when the benefits of hydrogels and electrospun nonwovens are combined.

TGF-β 1-induced IL-6 expression via MEK pathway in mesenchymal stem cells enhances NGF-dependent neurite extension in PC12 cells.

TGF-β 1-induced IL-6 expression via MEK pathway in mesenchymal stem cells enhances NGF-dependent neurite extension in PC12 cells.

Multichannel Bioactive Silk Nanofiber Conduits Direct and Enhance Axonal Regeneration after Spinal Cord Injury.

After a spinal cord injury, axonal regeneration over long distances is challenging due to the lack of physical guidance cues and bioactive signals. In this study, a multichannel bioactive silk fibroin nanofiber conduit was fabricated to improve spinal cord injury repair by enhancing axonal regeneration. The conduit was composed of longitudinally oriented silk fibroin nanofibers and then functionalized with laminin. In vitro, the bioactive conduits could promote neuron-like development and directional neurite extension of PC12 cells by providing a bioactive stimulus and physical guidance. In a spinal cord injury model in Sprague-Dawley rats, the biofunctionalized conduits displayed superior integration with the host tissue due to enhanced cell infiltration and tissue ingrowth. The glial scar was significantly reduced, allowing axonal ingrowth along with the channel direction. Compared to a single-channel conduit, the multichannel conduit improved spinal cord regeneration by boosting tissue ingrowth and axonal regeneration, indicating that the conduit architectures play critical roles in spinal cord regeneration. These silk fibroin conduits, along with the multichannel architecture, nanoscale cues, and the ability to bind bioactive compounds, represent promising candidates for spinal cord regeneration.

Ndr2 Kinase Controls Neurite Outgrowth and Dendritic Branching Through α1 Integrin Expression.

The serine/threonine kinase Ndr2 has been shown to control the inside-out activation of the β1subunit of integrins and the formation of neurites in both primary neurons and neurally differentiated pheochromacytoma (PC12) cells. In this study, we demonstrate that Ndr2 kinase furthermore determines the substrate specificity of neurite extension in PC12 cells via expression of α1β1 integrins. We show that stable overexpression of Ndr2 in PC12 cells increases neurite growth and extension on poly-D-lysine substrate, likely involving an increased expression of active β1 integrin in the growth tips of these cells. By contrast, the Ndr2 overexpressing cells do not show the α1β1 integrin-mediated enhancement of neurite growth on collagen IV substrate that can be seen in control cells. Moreover, they entirely fail to increase in response to activation of α1β1 integrins via a soluble KTS ligand and show a diminished accumulation of α1 integrin in neurite tips, although the expression of this subunit is induced during differentiation to comparable levels as in control cells. Finally, we demonstrate that Ndr2 overexpression similarly inhibits the α1β1 integrin-dependent dendritic growth of primary hippocampal neurons on laminin 111 substrate. By contrast, lack of Ndr2 impairs the dendritic growth regardless of the substrate. Together, these results suggest that Ndr2 regulates α1 integrin trafficking in addition to β1 integrin subunit activation and thereby controls the neurite growth on different extracellular matrix (ECM) substrates.

Conductive micropatterned polyurethane films as tissue engineering scaffolds for Schwann cells and PC12 cells

Controlling cellular alignment and elongation has been demonstrated as an important parameter for developing nerve tissue engineering scaffolds. Many approaches have been developed to guide cellular orientation for nerve regeneration such as micropatterning techniques. However, most of materials used for developing micropatterning scaffolds lack of bioactivity and biofunctionability. Here we present a functional conductive micropatterned scaffold based on bioactive conductive biodegradable polyurethane prepared using a micro-molding technique. These conductive micropatterned scaffolds are able to not only induce the Schwann cells (SCs) alignment and elongation by the micropatterned surface but also enhance the nerve growth factor (NGF) gene expression of SCs by the bioactivity of these materials. Additionally, the combined effect of the bioactivity of such conductive materials and the micropatterned structure also dramatically promotes the neurite extension and elongation of PC12 cells in a highly aligned direction. These data suggest that these conductive micropatterned scaffolds that easily control cellular orientation and organization, and dramatically enhance NGF gene expression and significantly induce the neurite extension of PC12 cells, have a great potential for nerve regeneration applications.

The Effect of Plasma Treated PLGA/MWCNTs-COOH Composite Nanofibers on Nerve Cell Behavior.

Electrospun nanofibrous scaffolds which can mimic the architecture of the natural extracellular matrix (ECM) are potential candidates for peripheral nerve repair application. Multi-walled carbon nanotubes (MWCNTs) are used in peripheral nerve repair due to their ability to promote neurite extension and support neural network formation. In this study, surface-modified nanofibrous scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and various ratios of carboxyl-modified MWCNTs (MWCNTs-COOH) (PC0, PC2, PC4 and PC8) were fabricated by electrospinning. The effects of MWCNTs-COOH on the fibers’ morphology, diameter distribution, mechanical properties and surface hydrophilicity were characterized by Scanning Electron Microscopy (SEM), ImageJ software, tensile testing and water contact angle. Furthermore, air plasma treatment was applied to improve the surface hydrophilicity of the scaffolds, and the optimal treatment condition was determined in terms of surface morphology, water contact angle and PC12 cell adhesion. Plasma treated nanofibers (p-PC0, p-PC2, p-PC4 and p-PC8) under optimal treatment conditions were used for further study. PC12 cell proliferation and differentiation were both improved by the addition of MWCNTs-COOH in scaffolds. Additionally, the proliferation and maturation of Schwann cells were enhanced on scaffolds containing MWCNTs-COOH. The neurite outgrowth of rat dorsal root ganglia (DRG) neurons was promoted on MWCNTs-COOH-containing scaffolds, and those cultured on p-PC8 scaffolds showed elongated neurites with a length up to 78.27 μm after 3 days culture. Our results suggested that plasma treated nanofibers under appropriate conditions were able to improve cell attachment. They also demonstrated that plasma treated scaffolds containing MWCNTs-COOH, especially the p-PC8 nanofibrous scaffold could support the proliferation, differentiation, maturation and neurite extension of PC12 cells, Schwann cells and DRG neurons. Therefore, p-PC8 could be a potential candidate for peripheral nerve regeneration application.

Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation.

Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits the differentiation of human neural progenitor cells and influences the expression of proteins associated with several cellular functions. We aimed to determine whether such chronic irradiation influences the expression of proteins associated with PC12 cells. Chronic irradiation at 0.027 mGy/min resulted in inhibition of NGF-induced neurite extension. Furthermore, irradiation enhanced the nerve growth factor (NGF)-induced increase in the phosphorylation of extracellular signal–regulated kinase (ERK), but did not affect the phosphorylation of NGF receptors, suggesting that irradiation influences pathways unassociated with the activation of ERK. We then examined whether irradiation influenced the Akt−Rac1 pathway, which is unaffected by ERK activation. Chronic irradiation also enhanced the NGF-induced increase in Akt phosphorylation, but markedly inhibited the NGF-induced increase in Rac1 activity that is associated with neurite extension. These results suggest that the inhibitory effect of irradiation on neurite extension influences pathways unassociated with Akt activation. As Ca2+/calmodulin-dependent kinase II (CaMKII) is known to inhibit the NGF-induced neurite extension in PC12 cells, independent of ERK and Akt activation, we next examined the effects of irradiation on CaMKII activation. Chronic irradiation induced CaMKII activation, while application of KN-62 (a specific inhibitor of CaMKII), attenuated increases in CaMKII activation and recovered neurite extension and NGF-induced increases in Rac1 activity that was inhibited by irradiation. Our results suggest that chronic irradiation with low-dose-rate γ-rays inhibits Rac1 activity via CaMKII activation, thereby inhibiting NGF-induced neurite extension.

Nanofibrous gelatine scaffolds integrated with nerve growth factor-loaded alginate microspheres for brain tissue engineering.

Neural regeneration research is designed in part to develop strategies for therapy after nerve damage due to injury or disease. In this study, a new gelatine-based biomimetic scaffold was fabricated for brain tissue engineering applications. A technique combining thermally induced phase separation and porogen leaching was used to create interconnected macropores and nanofibrous structure. To promote tissue regeneration processes, the scaffolds were integrated with nerve growth factor (NGF)-loaded alginate microspheres. The results showed that nanofibrous matrix could only be obtained when gelatine concentration was at least 7.5% (w/v). The scaffold with a modulus value (1.2kPa) similar to that of brain tissue (0.5-1kPa) was obtained by optimizing the heat treatment time, macropore size and gelatine concentration. The encapsulation efficiencies of NGF into 0.1% and 1% alginate microspheres were 85% and 100%, respectively. The release rate of NGF from the microspheres was controlled by the alginate concentration and the poly(L-lysine) coating. The immobilization of the microspheres in the scaffold reduced burst release and significantly extended the release period. The nanofibrous architecture and controlled release of NGF from the microspheres induced neurite extension of PC12 cells, demonstrating that the released NGF was in an active form. The results suggest that the scaffolds prepared in this study may have potential applications in brain tissue engineering due to topologic and mechanical properties similar to brain tissue and pore structure suitable for cell growth and differentiation. Copyright © 2016 John Wiley & Sons, Ltd.

Development of Novel 3-D Printed Scaffolds With Core-Shell Nanoparticles for Nerve Regeneration.

A traumatic injury of peripheral nerves is serious clinical problem that may lead to major loss of nerve function, affecting quality of patient's life. Currently, nerve autograft is widely used to reconstruct the nerve gap. However, such surgical procedure suffers from many disadvantages including donor site morbidity and limited availability. In order to address these issues, neural tissue engineering has focused on the development of synthetic nerve scaffolds to support bridging a larger gap and improving nerve generation. For this purpose, we fabricated a novel 3-D biomimetic scaffold, which has tunable porous structure and embedded core-shell nanoparticles with sustained neurogenic factor delivery system, using stereolithography based 3-D printing and coaxial electrospraying techniques. Our results showed that scaffolds with larger porosity significantly improve PC-12 neural cell adhesion compared to ones with smaller porosity. Furthermore, scaffolds embedded with bovine serum albumin containing nanoparticles showed an enhancement in cell proliferation relative to bared control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with nerve growth factor nanoparticles greatly increased the length of neurites and directed neurite extension of PC-12 cells along the fiber. In addition, the 3-D printed nanocomposite scaffolds also improved the average neurite length of primary cortical neurons. The results in this study demonstrate the potential of this 3-D printed scaffold in improving neural cell function and nerve growth.

C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity.

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2.

Development of multifunctional films for peripheral nerve regeneration.

This manuscript focuses on the development of multifunctional degradable polymer films that provide topographic cues for guided growth, surface gradients of growth factors as well as nanoparticles in the films for tunable release of growth factors to enable peripheral nerve regeneration. The combination of cues was designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. These multifunctional films successfully provided high protein loading capacities while persevering activity, protein gradients on the surface, and tunable release of bioactive nerve growth factor that promoted directional and guided neurite extension of PC12 cells of up to 10μm in 2days. These multifunctional films can be made into conduits for peripheral nerve regeneration.

X線照射はPC12細胞における神経成長因子誘導の神経軸索伸長を促進する

X線照射はPC12細胞における神経成長因子誘導の神経軸索伸長を促進する

Tenuifoliside A promotes neurite outgrowth in PC12 cells via the PI3K/AKT and MEK/ERK/CREB signaling pathways.

Previous studies have demonstrated the neuroprotective effect of tenuifoliside A (TFSA) on corticosterone-induced neuron damage in SH‑SY5Y cells, however, the effect of TFSA on the promotion of neurite outgrowth remains to be elucidated. PC12 cells were treated with TFSA or nerve growth factor, and the levels of proteins were evaluated by western blotting. In addition, for pharmacological experiments, inhibitors of the PD98059 mitogen‑activated protein kinase kinase (MEK) and phosphatidylinositol 3‑kinase (PI3K; LY294002) were added into the culture medium. The present study demonstrated that TFSA significantly increased the percentage of neurite‑bearing cells and promoted neurite extension in PC12 cells. In addition, TFSA‑treated PC12 cells also expressed increased levels of the 43 kD growth‑associated protein (GAP‑43) neural marker, comparable to those in nerve growth factor‑treated cells. The present study also demonstrated that TFSA enhanced the phosphorylation of extracellular signal‑regulated kinase (ERK) and Akt, which are important signaling molecules involved in neural differentiation in PC12 cells. Co‑treatment of the PC12 cells with the PD98059 MEK inhibitor and LY294002 PI3K inhibitor inhibited the neurite outgrowth induced by TFSA. In addition, treatment with TFSA also promoted the phosphorylation of cyclic AMP response element‑binding protein (CREB), which was inhibited completely by treatment with PD98059. In conclusion, the results of the present study demonstrated that TFSA induces neurite extension of PC12 cells and suggested that activation of the MEK/ERK/CREB and PI3K/Akt signaling pathways is involved in this process.

Neuronal Networks on Nanocellulose Scaffolds.

Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks.

Latent process genes for cell differentiation are common decoders of neurite extension length

A latent process involving signal transduction and gene expression is needed as a preparation step for cellular function. We previously found that nerve growth factor (NGF)-induced cell differentiation has a latent process, which is dependent on ERK activity and gene expression and required for subsequent neurite extension. A latent process can be considered as a preparation step that decodes extracellular stimulus information into cellular functions; however, molecular mechanisms of this process remain unknown. We identified Metrnl, Dclk1 and Serpinb1a as genes that are induced during the latent process (LP) with distinct temporal expression profiles and are required for subsequent neurite extension in PC12 cells. The LP genes showed distinct dependency on the duration of ERK activity, and they were also induced during the latent process of PACAP- and forskolin-induced cell differentiation. Regardless of neurotrophic factors, expression levels of the LP genes during the latent process (0-12 hours), but not phosphorylation levels of ERK, always correlated with subsequent neurite extension length (12-24 hours). Overexpression of all LP genes together, but not of each gene separately, enhanced NGF-induced neurite extension. The LP gene products showed distinct spatial localization. Thus, the LP genes appear to be the common decoders for neurite extension length regardless of neurotrophic factors, and they might function in distinct temporal and spatial manners during the latent process. Our findings provide molecular insight into the physiological meaning of the latent process as the preparation step for decoding information for future phenotypic change.

Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Cistanches Herba enhances learning and memory by inducing nerve growth factor

Nerve growth factor (NGF) has potent biological activities such as preventing neuronal death, promoting neurite outgrowth, supporting synapse formation, and enhancing memory function. NGF and NGF-like molecules can potentially be used to treat neurodegenerative disorders, such as dementia. This study investigated the effects of Cistanches Herba, a widely used medicinal herb, on NGF regulation and its neuronal actions, including neurite outgrowth, synapse formation, and learning and memory enhancement. Cistanches Herba extract (CHE; 250 μg/ml) increased NGF induction in C6 cells and led to neurite extension in PC12 cells. It also stimulated NGF secretion in the cortex and hippocampus of the mouse brain at 5 and 20 mg/kg/day (3 days, p.o.). Furthermore, CHE increased neuronal cell differentiation, neurite length, and synapse formation in the mouse hippocampus. CHE significantly enhanced learning and memory, as demonstrated by passive avoidance test and novel object recognition test. These results suggest that CHE is useful for improving memory function via its action in upregulating NGF.

Biocompatibility of biodegradable semiconducting melanin films for nerve tissue engineering

The advancement of tissue engineering is contingent upon the development and implementation of advanced biomaterials. Conductive polymers have demonstrated potential for use as a medium for electrical stimulation, which has shown to be beneficial in many regenerative medicine strategies including neural and cardiac tissue engineering. Melanins are naturally occurring pigments that have previously been shown to exhibit unique electrical properties. This study evaluates the potential use of melanin films as a semiconducting material for tissue engineering applications. Melanin thin films were produced by solution processing and the physical properties were characterized. Films were molecularly smooth with a roughness (Rms) of 0.341nm and a conductivity of 7.00±1.10×10−5Scm−1 in the hydrated state. In vitro biocompatibility was evaluated by Schwann cell attachment and growth as well as neurite extension in PC12 cells. In vivo histology was evaluated by examining the biomaterial–tissue response of melanin implants placed in close proximity to peripheral nerve tissue. Melanin thin films enhanced Schwann cell growth and neurite extension compared to collagen films in vitro. Melanin films induced an inflammation response that was comparable to silicone implants in vivo. Furthermore, melanin implants were significantly resorbed after 8 weeks. These results suggest that solution-processed melanin thin films have the potential for use as a biodegradable semiconducting biomaterial for use in tissue engineering applications.

Design and adsorption of modular engineered proteins to prepare customized, neuron-compatible coatings

Neural prosthetic implants are currently being developed for the treatment and study of both peripheral and central nervous system disorders. Effective integration of these devices upon implantation is a critical hurdle to achieving function. As a result, much attention has been directed towards the development of biocompatible coatings that prolong their in vivo lifespan. In this work, we present a novel approach to fabricate such coatings, which specifically involves the use of surface-adsorbed, nanoscale-designed protein polymers to prepare reproducible, customized surfaces. A nanoscale modular design strategy was employed to synthesize six engineered, recombinant proteins intended to mimic aspects of the extracellular matrix proteins fibronectin, laminin, and elastin as well as the cell–cell adhesive protein neural cell adhesion molecule. Physical adsorption isotherms were experimentally determined for these engineered proteins, allowing for direct calculation of the available ligand density present on coated surfaces. As confirmation that ligand density in these engineered systems impacts neuronal cell behavior, we demonstrate that increasing the density of fibronectin-derived RGD ligands on coated surfaces while maintaining uniform protein surface coverage results in enhanced neurite extension of PC-12 cells. Therefore, this engineered protein adsorption approach allows for the facile preparation of tunable, quantifiable, and reproducible surfaces for in vitro studies of cell–ligand interactions and for potential application as coatings on neural implants.