Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF.

Numerous signaling molecules are altered following nerve injury, serving as a blueprint for drug delivery approaches that promote nerve repair. However, challenges with achieving the appropriate temporal duration of recombinant protein delivery have limited the therapeutic success of this approach. Genetic engineering of mesenchymal stem cells (MSCs) to enhance the secretion of proangiogenic molecules such as vascular endothelial growth factor (VEGF) may provide an alternative. We hypothesized that the administration of VEGF-expressing human MSCs would stimulate neurite outgrowth and proliferation of cell-types involved in neural repair. When cultured with dorsal root ganglion (DRG) explants in vitro, control and VEGF-expressing MSCs (VEGF-MSCs) increased neurite extension and proliferation of Schwann cells (SCs) and endothelial cells, while VEGF-MSCs stimulated significantly greater proliferation of endothelial cells. When embedded within a 3D fibrin matrix, VEGF-MSCs maintained overexpression and expressed detectable levels over 21 days. After transplantation into a murine sciatic nerve injury model, VEGF-MSCs maintained high VEGF levels for 2 weeks. This study provides new insight into the role of VEGF on peripheral nerve injury and the viability of transplanted genetically engineered MSCs. The study aims to provide a framework for future studies with the ultimate goal of developing an improved therapy for nerve repair.

Redirection of neurite outgrowth by coupling chondroitin sulfate proteoglycans to polymer membranes.

Upon nerve injury, the body creates an environment consisting of permissive and non-permissive cues that instruct the function of cells involved in nerve repair. Among other roles, the developing extracellular matrix (ECM) acts as an underlying substrate to guide the union of neurites extending from the proximal stump for bridging the nerve gap. Chondroitin sulfate proteoglycans (CSPGs) are present in the nerve ECM and inhibit axon growth, potentially providing molecular cues to prevent aberrant growth and direct regeneration. In this study, we examined the potential of CSPGs to guide dorsal root ganglia (DRG) neurite outgrowth when freely available in the media or presented from a polymeric membrane. Soluble CSPGs added to the media of DRG explant cultures inhibited neurite outgrowth without spatial bias, caused retraction of axons, and decreased neurite extension in a dose-dependent manner. Poly-L-lactic acid membranes were chemically treated to enhance adsorption of CSPGs to the surface. CSPGs bound to 1,6-hexanediamine-treated membranes directed the orientation of neurite outgrowth, as neurites avoided bound CSPGs and a higher number and percentage grew on treated membranes lacking CSPGs. DRG explants cultured on CSPG-coated membranes without 1,6-hexanediamine-treatment had a smaller number of neurites and decreased neurite outgrowth, suggesting CSPGs were not retained on the membrane and were released into the culture medium. Taken together, these data demonstrate the potential of CSPG presentation to guide axonal growth. This approach offers a strategy to improve upon existing nerve guidance conduits by incorporating axon guidance molecules to direct nerve regeneration.

Neurite outgrowth in fibrin gels is regulated by substrate stiffness.

Fibrin is a promising matrix for use in promoting nerve repair given its natural occurrence in peripheral nerve injuries, and the biophysical properties of this matrix can be regulated to modulate tissue regeneration. In this study, we examined the effect of physical and mechanical properties of fibrin gels on dorsal root ganglia (DRG) neurite extension. Increases in fibrinogen concentration increased the number of fibrin strands, resulting in decreased pore size and increased stiffness. Neurite extension was reduced when DRG explants were cultured within fibrin gels of increasing fibrinogen concentrations (from 9.5 to 141 mg/mL). The addition of NaCl also increased the number of fibrin strands, reducing fiber diameter and porosity, while increasing mechanical strength, and reductions in neurite extension correlated with increases in NaCl content. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and matrix metalloproteinases by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction. Taken together, the results of this study provide new insight into the effect of fibrin gel biophysical properties on neurite extension and suggest new opportunities to improve the efficacy of these materials when used as nerve guidance conduits.

Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels.

Techniques that allow cells to self-assemble into three-dimensional (3-D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular--especially in fields such as stem cell research, tissue engineering, and cancer biology. Unfortunately, caveats involving scale, expense, geometry, and practicality have hindered the widespread adoption of these techniques. We present an easy-to-use, inexpensive, and scalable technology for production of complex-shaped, 3-D microtissues. Various primary cells and immortal cell lines were utilized to demonstrate that this technique is applicable to many cell types and highlight differences in their self-assembly phenomena. When seeded onto micromolded, nonadhesive agarose gels, cells settle into recesses, the architectures of which optimize the requisite cell-to-cell interactions for spontaneous self-assembly. With one pipeting step, we were able to create hundreds of uniform spheroids whose size was determined by seeding density. Multicellular tumor spheroids (MCTS) were assembled or grown from single cells, and their proliferation was quantified using a modified 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. Complex-shaped (e.g., honeycomb) microtissues of homogeneous or mixed cell populations can be easily produced, opening new possibilities for 3-D tissue culture.