At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)

In this paper the stimuli for and pattern of Schwann cell proliferation are defined under various experimental conditions . We used a tissue culture system in which fetal rat dorsal root ganglia, treated to eliminate contaminating fibroblasts (Wood, P ., 1976, Brain Res. 115 :361-375), appear to recapitulate many aspects of the developing peripheral nervous system . We observed that : (a) proliferation of Schwann cells on neurites is initially rapid, but, as each neurite becomes fully KEY WORDS Schwann cell proliferation " nerve tissue culture " autoradiography development . degeneration injury Peripheral nerve development is an ordered process (reviewed by Webster [61]) . The outgrowth of nerve fibers appears to be the initial event with naked nerve sprouts (lacking sheath cells) having been observed, for example, in the living tadpole tail fin (35,55,9) . Subsequently, Schwann cells of neural crest origin (35, 64) migrate along the nerve fibers and begin the process of ensheathment . At first, large groups of axons are surrounded by a few Schwann cells (45,42,28) . Subsequently, there is a burst of Schwann cell proliferation (57, 7) and invasion by the proliferating cells of the fascicles of naked axons (45, 21, among others) . Eventually, a sorting of axons occurs, with the largest axons (those destined to be myelinated) segregated into a 1 :1 relationship with the Schwann cells . Concomitant with the termination of Schwann cell proliferation, the myelin sheaths and connective tissue components of the peripheral nerve are formed.Cellular interactions between neurons and supporting cells are critical during development as well as subsequently (56,60) . To elucidate the nature of these interactions, a number of experi-J. CELL BIOLOGY

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Recent reports on the dystrophic mouse mutant suggest that the prominent extracellular matrix component of peripheral nerve tissues plays an important role in peripheral nerve development. We have examined the disposition of two prominent extracellular matrix components, fibronectin and laminin, both in mature peripheral nerve in vivo and in an in vitro system that allows study of Schwann cells in various functional states. In frozen sections of whole nerve, staining with antibodies to fibronectin and laminin shows that fibronectin stains throughout the endoneurium while laminin staining is restricted to regions known to contain basal lamina, particularly the basal lamina of each ensheathing Schwann cell. Tissue culture studies indicate that fibronectin staining at the light microscopic level is a reliable marker for fibroblasts (and not Schwann cells) in culture; conversely, antibodies to laminin stain components related to the Schwann cell surface but not components related to fibroblasts. Unexpectedly, Schwann cells in culture produce laminin at all stages in development, whether in contact with axons or not. As Schwann cells in culture begin to ensheathe axons, punctate regions of laminin on their surfaces become confluent. After ensheathment is completed, a continuous line of staining is found in the region of the Schwann cell basal lamina. It has been established that Schwann cells produce a basal lamina only when in contact with axons. Therefore, the production of laminin appears to be necessary but not sufficient for basal lamina formation. (5) and fibronectin (6, 7).We have now studied the appearance of fibronectin and the basal lamina component laminin, both in vivo and during development of peripheral nerve constituents in vitro. The role of the ECM in peripheral nerve development is poorly understood; studies on the dystrophic mouse mutant (8-10) and observations on Schwann cell/neuron cultures grown in defined medium (11) have demonstrated a correlation between abnormal ECM development and aberrant Schwann cell-axonal relationships. We report here immunohistochemical evidence that (i) both fibronectin and laminin are components of the mature peripheral nerve but they are localized in two distinct patterns; (ii) in tissue culture preparations, antibodies to each protein differentially recognize Schwann cells (laminin) and fibroblasts (fibronectin); and (iii) laminin, a known component of the ECM, is expressed by the Schwann cell prior to the development of a morphologically recognizable basal lamina. A preliminary report of this work has appeared (12