Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin⁺, GFAP⁺ and Nestin⁺ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.

Background: Although many nerve prostheses have been proposed in recent years, in the case of consistent loss of nervous tissue peripheral nerve injury is still a traumatic pathology that may impair patient's movements by interrupting his motor-sensory pathways. In the last few decades tissue engineering has opened the door to new approaches;: however most of them make use of rigid channel guides that may cause cell loss due to the lack of physiological local stresses exerted over the nervous tissue during patient's movement. Electrospinning technique makes it possible to spin microfiber and nanofiber flexible tubular scaffolds composed of a number of natural and synthetic components, showing high porosity and remarkable surface/volume ratio. Results: In this study we used electrospun tubes made of biodegradable polymers (a blend of PLGA/PCL) to regenerate a 10-mm nerve gap in a rat sciatic nerve in vivo. Experimental groups comprise lesioned animals (control group) and lesioned animals subjected to guide conduits implantated at the severed nerve stumps, where the tubular scaffolds are filled with saline solution. Four months after surgery, sciatic nerves failed to reconnect the two stumps of transected nerves in the control animal group. In most of the treated animals the electrospun tubes induced nervous regeneration and functional reconnection of the two severed sciatic nerve tracts. Myelination and collagen IV deposition have been detected in concurrence with regenerated fibers. No significant inflammatory response has been found. Neural tracers revealed the re-establishment of functional neuronal connections and evoked potential results showed the reinnervation of the target muscles in the majority of the treated animals. Conclusion: Corroborating previous works, this study indicates that electrospun tubes, with no additional biological coating or drug loading treatment, are promising scaffolds for functional nervous regeneration. They can be knitted in meshes and various frames depending on the cytoarchitecture of the tissue to be regenerated. The versatility of this technique gives room for further scaffold improvements, like tuning the mechanical properties of the tubular structure or providing biomimetic functionalization. Moreover, these guidance conduits can be loaded with various fillers like collagen, fibrin, or self-assembling peptide gels or loaded with neurotrophic factors and seeded with cells. Electrospun scaffolds can also be synthesized in different micro-architectures to regenerate lesions in other tissues like skin and bone.