•A fibronectin-rich ECM facilitates neurite outgrowth from ganglion explants.•Average neurite lengths on ECM are similar to a laminin-coated surface.•Fibroblasts on a micropatterned substrate assemble aligned ECM fibrils.•Micropatterned decellularized ECM controls the direction of neurite outgrowth.Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

•Sites of non-enzymatic glycation on fibronectin within a matrix were identified.•Our mass spectrometry–proteomics approach detected nine different species of AGEs.•Matrix assembly was stimulated by cell interactions with an AGE-modified matrix.•The mechanism of increased matrix assembly involves the AGE receptor (RAGE).Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM.

A nanometer thick, micron scale-patterned interface on a polymeric material directs fibroblast proliferation into a highly aligned, confluent cell monolayer. These cells assemble fibronectin extracellular matrix (ECM) fibrils that are aligned with the pattern, and matrix alignment on the synthetic polymer surface is maintained throughout a decellularization process. Biologic relevance of this ECM-synthetic material composite is illustrated by directing oriented neurite outgrowth in register with the aligned matrix fibrils.

In the mammary gland, the stromal extracellular matrix (ECM) undergoes dramatic changes during development and in tumorigenesis. For example, normal adult breast tissue is largely devoid of the ECM protein fibronectin (FN) whereas high FN levels have been detected in the stroma of breast tumors. FN is an established marker for epithelial-mesenchymal transition (EMT), which occurs during development and has been linked to cancer. During EMT, epithelial cell adhesion switches from cell-cell contacts to mainly cell-ECM interactions, raising the possibility that FN may have a role in promoting this transition. Using MCF-10A mammary epithelial cells, we show that exposure to exogenous FN induces an EMT response including upregulation of the EMT markers FN, Snail, N-cadherin, vimentin, the matrix metalloprotease MMP2, α-smooth muscle actin and phospho-Smad2, as well as acquisition of cell migratory behavior. FN-induced EMT depends on Src kinase and extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase signaling but not on the immediate early gene EGR-1. FN initiates EMT under serum-free conditions; this response is partially reversed by a transforming growth factor (TGF)β-neutralizing antibody, suggesting that FN enhances the effect of endogenous TGFβ. EMT marker expression is upregulated in cells on a fragment of FN containing the integrin-binding domain but not other domains. Differences in gene expression between FN and Matrigel are maintained with addition of a subthreshold level of TGFβ1. Together, these results show that cells interacting with FN are primed to respond to TGFβ. The ability of FN to induce EMT shows an active role for the stromal ECM in this process and supports the notion that the increased levels of FN observed in breast tumors facilitate tumorigenesis.

Templating of cell spreading and proliferation is described that yields confluent layers of cells aligned across an entire two-dimensional surface. The template is a reactive, two-component interface that is synthesized in three steps in nanometer thick, micron-scaled patterns on silicon and on several biomaterial polymers. In this method, a volatile zirconium alkoxide complex is first deposited at reduced pressure onto a surface pattern that is prepared by photolithography; the substrate is then heated to thermolyze the organic ligands to form surface-bound zirconium oxide patterns. The thickness of this oxide layer ranges from 10 to 70 nanometers, which is controlled by alkoxide complex deposition time. The oxide layer is treated with 1,4-butanediphosphonic acid to give a monolayer pattern whose composition and spatial conformity to the photolithographic mask are determined spectroscopically. NIH 3T3 fibroblasts and human bone marrow-derived mesenchymal stem cells attach and spread in alignment with the pattern without constraint by physical means or by arrays of cytophilic and cytophobic molecules. Cell alignment with the pattern is maintained as cells grow to form a confluent monolayer across the entire substrate surface.

Tissue formation and cell differentiation depend on a properly assembled extracellular matrix (ECM). Fibronectin is a key constituent of the pericellular ECM, forming essential connections between cell surface integrin receptors and structural components of the ECM. Recent studies using vertebrate models, conditional gene knockouts, tissue explants, and cell culture systems have identified developmental processes that depend on fibronectin and its receptor α5β1 integrin. We describe requirements for fibronectin matrix in the cardiovascular system, somite and precartilage development, and epithelial–mesenchymal transition. Information about molecular mechanisms shows the importance of fibronectin and integrins during tissue morphogenesis and cell differentiation, as well as their cooperation with growth factors to mediate changes in cell behaviors.

Integrin receptors for extracellular matrix (ECM) are critical determinants of biological processes. Regulation of integrin expression is one way for cells to respond to changes in the ECM, to integrate intracellular signals, and to obtain appropriate adhesion for cell motility, proliferation, and differentiation. Transcriptional and post-translational mechanisms for changing the integrin repertoire at the cell surface have recently been described. These mechanisms work through transcriptional regulation that alters the proportions of one integrin relative to another, referred to as integrin switching, or through localized regulation of integrin–ECM interactions, thus providing exquisite control over cell rearrangements during tissue morphogenesis and remodeling. These integrin regulatory pathways may also be important targets in such emerging fields as tissue engineering and regenerative medicine.

Templating of cell spreading and proliferation is described that yields confluent layers of cells aligned across an entire two-dimensional surface. The template is a reactive, two-component interface that is synthesized in three steps in nanometer thick, micron-scaled patterns on silicon and on several biomaterial polymers. In this method, a volatile zirconium alkoxide complex is first deposited at reduced pressure onto a surface pattern that is prepared by photolithography; the substrate is then heated to thermolyze the organic ligands to form surface-bound zirconium oxide patterns. The thickness of this oxide layer ranges from 10 to 70 nanometers, which is controlled by alkoxide complex deposition time. The oxide layer is treated with 1,4-butanediphosphonic acid to give a monolayer pattern whose composition and spatial conformity to the photolithographic mask are determined spectroscopically. NIH 3T3 fibroblasts and human bone marrow-derived mesenchymal stem cells attach and spread in alignment with the pattern without constraint by physical means or by arrays of cytophilic and cytophobic molecules. Cell alignment with the pattern is maintained as cells grow to form a confluent monolayer across the entire substrate surface.

A nanometer thick, micron scale-patterned interface on a polymeric material directs fibroblast proliferation into a highly aligned, confluent cell monolayer. These cells assemble fibronectin extracellular matrix (ECM) fibrils that are aligned with the pattern, and matrix alignment on the synthetic polymer surface is maintained throughout a decellularization process. Biologic relevance of this ECM-synthetic material composite is illustrated by directing oriented neurite outgrowth in register with the aligned matrix fibrils.