Development of a scaffoldless three-dimensional engineered nerve using a nerve-fibroblast co-culture

Abstract

Nerve grafts are often required to replace tissue damaged by disease, surgery, or extensive trauma. Limitations such as graft availability, donor site morbidity, and immune rejection have led investigators to develop strategies to engineer nerve tissue. The goal of this study was to fabricate a scaffoldless three-dimensional (3D) nerve construct using a co-culture of fetal nerve cells with a fibroblast monolayer and allow the co-culture to remodel into a 3D construct with an external fibroblast layer and an internal core of interconnected neuronal cells. Primary fibroblasts were seeded on laminin-coated plates and allowed to form a confluent monolayer. Neural cells isolated from E-15 spinal cords were seeded on top of the fibroblast monolayer and allowed to form a networked monolayer across the monolayer of fibroblasts. Media shifts initiated contraction of the fibroblast monolayer and a remodeling of the co-culture into a 3D construct held statically in place by the two constraint pins. Immunohistochemistry using S100 (Schwann cell), beta3-tubulin, DAPI, and collagen I indicated an inner core of nerve cells surrounded by an external layer of fibroblasts. Conduction velocities of the 3D nerve and control (fibroblast-only) constructs were measured in vitro and compared to in vivo measures of neonatal sciatic nerve. The conduction velocities of the nerve constructs were comparable to 24-d-old neonatal nerve. The presence of Schwann cells and the ability to conduct neuronal signals in vitro suggest the scaffoldless 3D nerve constructs will be a viable option for nerve repair.

Figure 1

The effect of media on the formation of a confluent monolayer of fibroblasts. Fibroblasts were seeded a density of 1×10⁵ cells per plate and viewed 7 d after proliferation of laminin-coated sylgaard plated and fed with (A) GMA, (B) 50% GMA/50% NBM, (C) NBM, and (D) NBM for 5 d then GMA. Images were viewed at ×50.

Figure 2

The effect of co-culturing E-15 neural cells on an established monolayer of fibroblasts. (A) Neural cells on fibroblast monolayer after 3 d on neural basal medium, (B) neural cells on fibroblast monolayer stained with S100 and DAPI, and (C) neural cells on fibroblast monolayer after 7 d on neural basal medium formed a confluent network. Notice the decreased presence of fibroblasts. Images A and B are viewed at ×100. Image C was viewed at ×50.

Figure 3

Immunohistological characterization of 3-D nerve-fibroblast construct stained with (A) S100 and DAPI, (B) S100, DAPI, and collagen 1, and (C, D) β3-tubulin and DAPI. Images viewed at ×50.

Figure 4

Nerve conduction velocities of native sciatic nerve ranging from 1 to 4 wk old compared to measures taken of adult (12 wk old) native sciatic nerve and in vivo measures of nerve-only constructs and fibroblast-only constructs. Data represent mean ± standard deviations.