Schwann cell behavior in three-dimensional collagen gels: evidence for differential mechano-transduction and the influence of TGF-beta 1 in morphological polarization and differentiation.

Schwann cells (SCs) cultured on and within magnetically aligned collagen gels were examined for their abilities to spread and exhibit contact guidance, two functions that are relevant to their potential enhancement of neurite migration and regeneration in entubulation repair of transection-type nerve injuries. Cells seeded at or near the surfaces of gels abandoned their initially spherical shapes, adopting spread morphologies rapidly compared to cells within the gels. Those few cells within the gels that did spread exhibited marked contact guidance responses, aligning strongly with the aligned collagen fibrils. Spreading of cells in gels could not be induced by varied cell concentration, collagen density, mitogen presence, inclusion of soluble laminin, or use of fibrin gel in lieu of collagen. However, cells that settled at the interface between collagen gel layers during gellation of the top layer above a preformed bottom layer were highly spread. This suggests that a differential mechanical interaction across the cell at an interface, where at least one surface presents constituents of the basal lamina, permits the Schwann cell to rapidly revert to a spread, differentiated phenotype. Unlike other reagents, TGF-beta1 was able to induce significant SC spreading as early as 4 h post-seeding. Consistent with the differential-mechanical cue mechanism, TGF-beta1 appears to facilitate this response, at least in part, by upregulating beta1 integrin expression, thereby enabling the SC to more acutely detect these local cues in the mechanical environment.

Rational design of contact guiding, neurotrophic matrices for peripheral nerve regeneration.

Nerve guides filled with magnetically aligned hydrated gels of type I collagen have been shown to impart strong contact guidance cues to elongating neurites in vitro and to increase the number of regenerating axons in vivo relative to an isotropic collagen gel. We have formulated and analyzed a model to determine the conditions under which the target concentration of nerve growth factor (NGF) to support axonal growth can be sustained by entrapping either NGF-secreting cells or NGF-releasing polymer microspheres in the aligned gel. The equation describing NGF concentration with a distributed source term was solved after experimental determination of (1) the rate of NGF release from PLGA 85/15 microspheres, (2) the NGF diffusion coefficient in the gel and nerve guide membrane containing the gel, and (3) the maximum microsphere loading that does not compromise the magnetic alignment of collagen fibrils. We find that for a rat sciatic nerve, when using a 1 mm diameter nerve guide within a commercially available collagen membrane, the microsphere loading limit will prevent the construct's capacity to sustain the target NGF concentration of 1 ng/ml at two months when either wild type Schwann cells or PLGA 85/15 microspheres are used as the NGF source. This target concentration, however, will be maintained when transfected cells described in the literature to hypersecrete NGF are used, or when the microspheres are used if the permeability of the nerve guide membrane can be moderately decreased. For a human median nerve, when using a 5 mm diameter nerve guide within a commercially available membrane, the microspheres are capable of sustaining NGF concentrations above 1 ng/ml to at least 75 days without the need to decrease membrane permeability.