Steric Interference of Adhesion Supports In-Vitro Chondrogenesis of Mesenchymal Stem Cells on Hydrogels for Cartilage Repair.

Recent studies suggest the presence of cell adhesion motifs found in structural proteins can inhibit chondrogenesis. In this context, the current study aims to determine if a polyethylene glycol (PEG)-modified fibrinogen matrix could support better chondrogenesis of human bone marrow mesenchymal stem cells (BM-MSC) based on steric interference of adhesion, when compared to a natural fibrin matrix. Hydrogels used as substrates for two-dimensional (2D) BM-MSC cultures under chondrogenic conditions were made from cross-linked PEG-fibrinogen (PF) and compared to thrombin-activated fibrin. Cell morphology, protein expression, DNA and sulfated proteoglycan (GAG) content were correlated to substrate properties such as stiffness and adhesiveness. Cell aggregation and chondrogenic markers, including collagen II and aggrecan, were observed on all PF substrates but not on fibrin. Shielding fibrinogen's adhesion domains and increasing stiffness of the material are likely contributing factors that cause the BM-MSCs to display a more chondrogenic phenotype. One composition of PF corresponding to GelrinC™--a product cleared in the EU for cartilage repair--was found to be optimal for supporting chondrogenic differentiation of BM-MSC while minimizing hypertrophy (collagen X). These findings suggest that semi-synthetic biomaterials based on ECM proteins can be designed to favourably affect BM-MSC towards repair processes involving chondrogenesis.

The role of matrix metalloproteinases in regulating neuronal and nonneuronal cell invasion into PEGylated fibrinogen hydrogels.

Injured peripheral nerve tissue could benefit from biomaterial nerve guidance conduits (NGCs) that are designed to promote neuronal regeneration. Nerve regeneration is a complex multi-step process that involves the remodeling of the ECM surrounding the regenerating neural tissue. Hydrogel biomaterials have been used as provisional matrices to regulate this regeneration process by providing the desired physical properties and controllable degradation characteristics. The purpose of this investigation was to understand the mechanism by which nerve cells penetrate into a hydrogel made from PEGylated fibrinogen. In this context, the dorsal root ganglion (DRG) assay was used as an in vitro model to study the cellular invasion behavior of both neural and nonneuronal cells. Our hypothesis stipulated that DRG cells employ matrix metalloproteinases (MMPs) in order to degrade the dense hydrogel matrix and penetrate the biomaterial. Three dimensional (3D) DRG-hydrogel constructs were cultured with MMP inhibitors (MMPi) and the effect of the inhibitors on DRG cell outgrowth was investigated. We also examined the effect of inhibitors on two dimensional (2D) DRG cell outgrowth on PEGylated fibrinogen hydrogels and on tissue culture polystyrene (TCP). Our results demonstrate that DRG cell outgrowth into and onto PEGylated fibrinogen hydrogels was inhibited by MMPi and that the outgrowth characteristics was dependent on the type of inhibitor and its concentration. MMP-3i and MMP-8i decreased both neuronal and nonneuronal outgrowth, where MMP-3i had a stronger inhibitory effect on nonneuronal cells. MMP-2/9i, on the other hand, affected the neuronal outgrowth much more than the others. We concluded that MMPs play a central role in the process of DRG cell penetration into PEGylated fibrinogen hydrogels and may also regulate the adhesion, migration and elongation of neuronal cells on the surface of these hydrogel biomaterials.

Laser photoablation of guidance microchannels into hydrogels directs cell growth in three dimensions.

Recent years have seen rapid progress in the engineering and application of biomaterials with controlled biological, physical, and chemical properties, and the development of associated methods for micropatterning of three-dimensional tissue-engineering scaffolds. A remaining challenge is the development of robust, flexible methods that can be used to create physical guidance structures in cell-seeded scaffolds independently of environmental constraints. Here we demonstrate that focal photoablation caused by pulsed lasers can generate guidance structures in transparent hydrogels, with feature control down to the micron scale. These photopatterned microchannels guide the directional growth of neurites from dorsal root ganglia. We characterize the effect of laser properties and biomaterial properties on microchannel formation in PEGylated fibrinogen hydrogels, and the effect of photoablation on neural outgrowth. This strategy could lead to the development of a new generation of guidance channels for treating nerve injuries, and the engineering of structured three-dimensional neuronal or nonneuronal networks.

Compositional alterations of fibrin-based materials for regulating in vitro neural outgrowth.

The purpose of this investigation was to evaluate dorsal root ganglion (DRG) cell outgrowth in a biosynthetic material made from PEGylated fibrinogen and in thrombin-polymerized fibrin hydrogels. The hypothesis stipulated that changing the composition of the biosynthetic constituents of the hydrogel can be used to better regulate the rate of DRG cell invasion when compared to reconstituted fibrin hydrogels. The composition of the biosynthetic hydrogel was controlled by the relative amount of poly(ethylene glycol) (PEG) to fibrinogen or by the fibrinogen concentration in the material. Other PEGylated protein hydrogels including PEGylated collagen and PEGylated albumin were used as positive and negative controls, respectively, for DRG cell outgrowth studies. The PEGylated fibrinogen hydrogels readily supported extensive outgrowth of sprouting neurites and invading glial cells from encapsulated DRGs in the presence of nerve growth factor. The PEGylated fibrinogen hydrogels exhibited preferential neurite invasion whereby the nonneuronal cells were observed lagging behind the invading neurites. Hydrogels containing more PEG or fibrinogen reduced the lag between the cell types and also the overall rate of the cellular invasion. Time-lapse video microscopy and quantitative cellular invasion data were used to illustrate the effectiveness of controlling DRG cell outgrowth characteristics in PEGylated fibrinogen hydrogels when compared to their respective reconstituted protein hydrogel counterparts.

A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination.

Myelination in the peripheral nervous system requires close contact between Schwann cells and the axon, but the underlying molecular basis remains largely unknown. Here we show that cell adhesion molecules (CAMs) of the nectin-like (Necl, also known as SynCAM or Cadm) family mediate Schwann cell-axon interaction during myelination. Necl4 is the main Necl expressed by myelinating Schwann cells and is located along the internodes in direct apposition to Necl1, which is localized on axons. Necl4 serves as the glial binding partner for axonal Necl1, and the interaction between these two CAMs mediates Schwann cell adhesion. The disruption of the interaction between Necl1 and Necl4 by their soluble extracellular domains, or the expression of a dominant-negative Necl4 in Schwann cells, inhibits myelination. These results suggest that Necl proteins are important for mediating axon-glia contact during myelination in peripheral nerves.

Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier.

Accumulation of Na(+) channels at the nodes of Ranvier is a prerequisite for saltatory conduction. In peripheral nerves, clustering of these channels along the axolemma is regulated by myelinating Schwann cells through a yet unknown mechanism. We report the identification of gliomedin, a glial ligand for neurofascin and NrCAM, two axonal immunoglobulin cell adhesion molecules that are associated with Na+ channels at the nodes of Ranvier. Gliomedin is expressed by myelinating Schwann cells and accumulates at the edges of each myelin segment during development, where it aligns with the forming nodes. Eliminating the expression of gliomedin by RNAi, or the addition of a soluble extracellular domain of neurofascin to myelinating cultures, which caused the redistribution of gliomedin along the internodes, abolished node formation. Furthermore, a soluble gliomedin induced nodal-like clusters of Na+ channels in the absence of Schwann cells. We propose that gliomedin provides a glial cue for the formation of peripheral nodes of Ranvier.