Spontaneous Helical Structure Formation in Laminin Nanofibers.
ABSTRACT: Laminin is a cross-shaped heterotrimer composed of three polypeptides chains that assembles into an insoluble extracellular matrix (ECM) network as part of the basement membrane, serving a vital role in many processes such as embryonic development, differentiation, and muscle and nerve regeneration. Here we engineered monodisperse laminin nanofibers using a surface-initiated assembly technique in order to investigate how changes in protein composition affect formation and structure of the network. Specifically, we compared laminin 111 with varying degrees of purity and with and without entactin to determine whether these changes alter biophysical properties. All the laminin types were reproducibly patterned as 200 ?m long, 20 ?m wide nanofibers that were successfuly released during surface-initiated assembly into solution. All nanofibers contracted upon release, and while initial lengths were identical, lengths of released fibers depended on the laminin type. Uniquely, the laminin 111 at high purity (>95%) and without entactin spontaneouly formed helical nanofibers at greater than 90%. Atomic force microscopy revealed that the nanofiber contraction was associated with a change in nanostructure from fibrillar to nodular, suggestive of refolding of laminin molecules into a globular-like conformation. Further, for the high purity laminin that formed helices, the density of the laminin at the edges of the nanofiber was higher than in the middle, providing a possible origin for the differential pre-stress driving the helix formation. Together, these results show that variation in the purity of laminin 111 and presence of entactin can have significant impact on the biophysical properties of the assembled protein networks. This highlights the fact that our understanding of protein assembly and function is still incomplete and that cell-free, in vitro assays can provide unique insights into the ECM.
Project description:<h4>Purpose</h4>To study the composition of the internal limiting membrane (ILM) of the retina, the extracellular matrix (ECM) of idiopathic epiretinal membranes (iERMs), and the relationships occurring between the two membranes.<h4>Methods</h4>Forty-six iERMs, 24 of them associated with the ILM, were collected and included in this study. The investigation has been carried out by immunofluorescence and confocal microscopy on glutaraldehyde- and osmium-fixed epon-embedded samples and on frozen samples. Sections were double or triple labelled with antibodies against vimentin; collagens I, III, IV, α5(IV), and VI; laminin 1 + 2; laminin α2-, α4-, α5-, β1-, β2-, β3-, γ1-, and γ2-chains; entactin; and fibronectin.<h4>Results</h4>iERM thickness was not uniform. Almost 14% of iERMs showed thickenings due to folding of their ECM component under the cell layer. The vitreal side of iERMs was often shorter than the attached ILM. In this case, the ILM resulted folded under the iERM. ILMs contained laminin 111; laminin α2-, α5-, β1-, β2-, and γ1-chains; entactin; collagens I; α5(IV); [α1(IV)]<sub>2</sub>α2(IV); and VI. Laminins, entactin, and α5(IV) were gathered on the retinal half of the ILM, whereas collagens [α1(IV)]<sub>2</sub>α2(IV) and I were restricted to the vitreal side. Collagen VI was detected on both sides of the ILM. iERMs expressed laminin 111, collagens III, [α1(IV)]<sub>2</sub>α2(IV) and VI, entactin, and fibronectin. Entactin co-localized with laminins and collagen IV.<h4>Conclusions</h4>Analysis of laminins and collagen chain expression indicates that ILM contains laminin 111 (former laminin 1), laminin 521 (former laminin 11), laminin 211 (former laminin 2), collagen [α1(IV)]<sub>2</sub>α2(IV), and collagen α3(IV)α4(IV)α5. In contrast, iERMs express only collagen [α1(IV)]<sub>2</sub>α2(IV) and laminin 111. In addition, both iERMs and ILMs contain entactin. The presence of three major constituents of the basement membranes co-localized together in iERMs is suggestive for a deranged process of basement membrane formation which fails to assemble properly. In view of the many interactions occurring among its proteins, the ECM of either the iERMs or the ILMs can account for their reciprocal adhesiveness. In addition, the peculiar deposition of the ECM observed in some samples of iERM is suggestive for its involvement in the formation of macular puckers.
Project description:Epithelial cell types typically lose apicobasal polarity when cultured on 2D substrates, but apicobasal polarity is required for directional secretion by secretory cells, such as salivary gland acinar cells. We cultured salivary gland epithelial cells on poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds that mimic the basement membrane, a specialized extracellular matrix, and examined cell proliferation and apicobasal polarization. Although cells proliferated on nanofibers, chitosan-coated nanofiber scaffolds stimulated proliferation of salivary gland epithelial cells. Although apicobasal cell polarity was promoted by the nanofiber scaffolds relative to flat surfaces, as determined by the apical localization of ZO-1, it was antagonized by the presence of chitosan. Neither salivary gland acinar nor ductal cells fully polarized on the nanofiber scaffolds, as determined by the homogenous membrane distribution of the mature tight junction marker, occludin. However, nanofiber scaffolds chemically functionalized with the basement membrane protein, laminin-111, promoted more mature tight junctions, as determined by apical localization of occludin, but did not affect cell proliferation. To emulate the multifunctional capabilities of the basement membrane, bifunctional PLGA nanofibers were generated. Both acinar and ductal cell lines responded to signals provided by bifunctional scaffolds coupled to chitosan and laminin-111, demonstrating the applicability of such scaffolds for epithelial cell types.
Project description:Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells' growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, "IKVAV", and fibronectin-derived well known adhesive sequence, "RGD", into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.
Project description:Optimization of nanofiber surface properties can lead to enhanced tissue regeneration outcomes in the context of bone tissue engineering. Herein, we developed a facile strategy to decorate elctrospun nanofibers using extracellular matrix (ECM) in order to improve their performance for bone tissue engineering. Electrospun PLLA nanofibers (PLLA NF) were seeded with MC3T3-E1 cells and allowed to grow for two weeks in order to harvest a layer of ECM on nanofiber surface. After decellularization, we found that ECM was successfully preserved on nanofiber surface while maintaining the nanostructure of electrospun fibers. ECM decorated on PLLA NF is biologically active, as evidenced by its ability to enhance mouse bone marrow stromal cells (mBMSCs) adhesion, support cell proliferation and promote early stage osteogenic differentiation of mBMSCs. Compared to PLLA NF without ECM, mBMSCs grown on ECM/PLLA NF exhibited a healthier morphology, faster proliferation profile, and more robust osteogenic differentiation. Therefore, our study suggests that ECM decoration on electrospun nanofibers could serve as an efficient approach to improving their performance for bone tissue engineering.
Project description:The accumulation of extracellular matrix (ECM) and glial scar formation are considered important factors for the failure of regeneration in central nervous system (CNS) injury and multiple sclerosis. Theiler's murine encephalomyelitis (TME) as a model of multiple sclerosis served to evaluate the spatio-temporal course of ECM alterations in demyelinating conditions. Microarray analysis revealed only mildly upregulated gene expression of ECM molecules, their biosynthesis pathways and pro-fibrotic factors, while upregulation of matrix remodeling enzymes was more prominent. Immunohistochemistry demonstrated progressive accumulation of chondroitin sulfate proteoglycans, glycoproteins and collagens within demyelinated TME lesions, paralleling the development of astrogliosis. Deposition of collagen IV, laminin, perlecan and tenascin-C started 28 days postinfection (dpi), collagen I, decorin, entactin and neurocan accumulated from 56 dpi on, and fibronectin from 98 dpi on. The basement membrane (BM) molecules collagen IV, entactin, fibronectin, laminin and perlecan showed perivascular and parenchymal deposition, while the non-BM components collagen I, decorin, neurocan and tenascin-C only accumulated in a nonvascular pattern in demyelinated areas. Contrary, phosphacan expression progressively decreased during TME. The immunoreactivity of aggrecan and brevican remained unchanged. The spatio-temporal association of matrix accumulation with astrogliosis suggests a mainly astrocytic origin of ECM deposits, which in turn may contribute to remyelination failure in TME.
Project description:Peptide nanofibers are useful for many biological applications, including immunotherapy, tissue engineering, and drug delivery. The robust lengthwise assembly of these peptides into nanofibers is typically difficult to control, resulting in polydisperse fiber lengths and an incomplete understanding of how nanofiber length affects biological responses. Here, rationally designed capping peptides control the length of helical peptide nanofibers with unique precision. These designed peptides bind the tips of elongated nanofibers to shorten and narrow their length distributions. Demonstrating their use as immunotherapies, capped nanofibers are preferentially cross-presented by dendritic cells compared to uncapped nanofibers. Due to increased cross-presentation, these capped nanofibers trigger stronger CD8<sup>+</sup> T-cell responses in mice than uncapped nanofibers. This strategy illustrates a means for controlling the length of supramolecular peptide nanofibers to modulate their immunogenicity in the context of immunotherapies.
Project description:In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence-like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.
Project description:We recently reported simultaneous self-assembly, alignment, and patterning of peptide amphiphile (PA) nanofibers over large areas by a soft lithographic technique termed sonication-assisted solution embossing (SASE). The present work examines the effect of ultrasonication, channel width, and nanofiber persistence length lambda on the degree of alignment of nanofibers patterned by SASE. Polarized transmission and reflection infrared spectroscopy are used to establish a figure of merit for comparing nanofiber alignment based on a model of the supramolecular structures being composed of oriented beta-sheets. The aligned nanostructures show orientation parameters of up to 0.4, and estimates of the persistence length lambda from atomic force microscopy (AFM) images range from 2.0 to 11 mum depending on the chemical structure of molecules used. The data suggest that stiffer nanofibers, defined as those with longer persistence lengths, may actually align less well due to increased difficulty in cleaving them during the alignment process. Alignment can be enhanced with the addition of ultrasonic agitation and confinement of the self-assembled structures within channels around 400 nm in width.
Project description:The extracellular matrix (ECM) is a fibrillar protein-based network, the physical and chemical properties of which can influence a multitude of cellular processes. Despite having an important role in cell and tissue signaling, a complete chemo-mechanical characterization of ECM proteins such as fibronectin (FN) is lacking. In this study, we engineered monodisperse FN nanofibers using a surface-initiated assembly technique in order to provide new insight into the elastic behavior of this material over large deformations. FN nanofibers were patterned on surfaces in a pre-stressed state and when released from the surface underwent rapid contraction. We found that the FN nanofibers underwent 3.3-fold and 9-fold changes in length and width, respectively, and that the nanofiber volume was conserved. Volume was also conserved following uniaxial extension of the FN nanofibers of ~2-fold relative to the patterned state. This data suggests that the FN networks we engineered formed an incompressible material with a Poisson's ratio of ~0.5. While the Poisson's ratio of cells and other biological materials are widely estimated as 0.5, our experimental results demonstrate that for FN networks this is a reasonable approximation.
Project description:Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the ?1 chain from Laminin-111 and the YIGSR peptide corresponding to the ?1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.