Rapidly formed stable and aligned dense collagen gels seeded with Schwann cells support peripheral nerve regeneration.

OBJECTIVE: Gel aspiration-ejection (GAE) has recently been developed for the rapid production of dense, anisotropic collagen gel scaffolds with adjustable collagen fibrillar densities. In this study, a GAE system was applied to produce aligned Schwann cells within a type-1 collagen matrix to generate GAE-engineered neural tissues (GAE-EngNT) for potential nerve tissue engineering applications. APPROACH: The stability and mechanical properties of the constructs were investigated along with the viability, morphology and distribution of Schwann cells. Having established the methodology to construct stable robust Schwann cell-loaded engineered neural tissues using GAE (GAE-EngNTs), the potential of these constructs in supporting and guiding neuronal regeneration, was assessed both in vitro and in vivo. MAIN RESULTS: Dynamic mechanical analysis strain and frequency sweeps revealed that the GAE-EngNT produced via cannula gauge number 16 G (∼1.2 mm diameter) exhibited similar linear viscoelastic behaviors to rat sciatic nerves. The viability and alignment of seeded Schwann cells in GAE-EngNT were maintained over time post GAE, supporting and guiding neuronal growth in vitro with an optimal cell density of 2.0 × 106 cells ml-1. An in vivo test of the GAE-EngNTs implanted within silicone conduits to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that the constructs significantly promoted axonal regeneration and vascularization across the gap, as compared with the empty conduits although less effective regeneration compared with the autograft groups. SIGNIFICANCE: Therefore, this is a promising approach for generating anisotropic and robust engineered tissue which can be used with Schwann cells for peripheral nerve repair.

Mechanical Response of Neural Cells to Physiologically Relevant Stiffness Gradients.

Understanding the influence of the mechanical environment on neurite behavior is crucial in the development of peripheral nerve repair solutions, and could help tissue engineers to direct and guide regeneration. In this study, a new protocol to fabricate physiologically relevant hydrogel substrates with controlled mechanical cues is proposed. These hydrogels allow the analysis of the relative effects of both the absolute stiffness value and the local stiffness gradient on neural cell behavior, particularly for low stiffness values (1-2 kPa). NG108-15 neural cell behavior is studied using well-characterized collagen gradient substrates with stiffness values ranging from 1 to 10 kPa and gradient slopes of either 0.84 or 7.9 kPa mm-1 . It is found that cell orientation is influenced by specific combinations of stiffness value and stiffness gradient. The results highlight the importance of considering the type of hydrogel as well as both the absolute value of the stiffness and the steepness of its gradient, thus introducing a new framework for the development of tissue engineered scaffolds and the study of substrate stiffness.

Physical and mechanical properties of RAFT-stabilised collagen gels for tissue engineering applications.

Cell behavior is influenced by the mechanical and structural properties of their substrate environment. Also, materials mechanically resistant to surgical handling and similar to the host site are required in tissue engineering to minimise the chance of an adverse host response. RAFT-stabilisation is a commercially available technique for creating stabilised hydrogels. Properties of RAFT-stabilised collagen (RsC) gels are governed by size, composition and arrangement of fibrils and their interaction with the fluid trapped within the matrix. The stabilisation process, using hydrophilic porous absorbers, produces dense matrices by rapid expulsion of fluid, and the structure obtained has mechanical properties suitable for tissue engineering. However, protocols to define and compare the physical properties and mechanical behavior of RAFT-stabilised collagen gels are not standardised across the field. Here, we investigate the fundamental mechanical and structural properties of RsC gels, and propose a new empirical relationship that correlates the measured stiffness of gels to varying frequency of strain oscillation. The results provide quantitative data characterising this extracellular environment for future tissue engineering studies.