Nerve fibroblast impact on Schwann cell behavior.

In order to reveal non-neuronal cell interactions after peripheral nerve lesions, we began to analyze the impact of sciatic nerve fibroblasts on Schwann cells in vitro. Both cell types are considered to have opposite effects on axonal regeneration. Few data are available on how repulsive nerve fibroblasts affect neuritotrophic Schwann cells and thus might indirectly influence axonal regrowth. Using different culture systems in conjunction with time-lapse video recording, metabolic labeling, pharmacological intervention, RNAi knockdown, Western blotting and RT-PCR analysis, we found that nerve fibroblasts differentially modify the various responses of Schwann cells. In the presence of collagen type IV and heparan sulfate proteoglycan but not of laminin, diffusible fibroblast factors slow down Schwann cell proliferation. In contrast, fibroblast factors increase the migratory activity of Schwann cells without being chemoattractive. One pro-migratory fibroblast factor turned out to be neuregulin. The pro-migratory activity of nerve fibroblasts and of recombinant neuregulin-1beta1 can be counteracted by neuregulin-specific pharmacological intervention and by neuregulin RNA interference. We show for the first time that nerve fibroblasts play antagonistic and agonistic roles for Schwann cells in a context-dependent manner. The data shed light on cellular mechanisms and have implications for some neuro-tissue engineering strategies.

The pro-angiogenic characteristics of a cross-linked gelatin matrix.

To overcome limitations on regeneration in the nervous system and other organs caused by insufficient blood supply, we have developed a gelatin sponge material which stimulates blood vessel formation, i.e. angiogenesis. Controlled chemical cross-linking was employed to slow down enzymatic degradation of the gelatin matrix. Four different in vitro assays using L929 fibroblasts and purified endothelial cells indicated that the sponge material did not release toxic components, but provided a permissive substratum for cell attachment, cell migration and pronounced cell proliferation, all of which are crucial for the formation of vasculature. Two in vivo models were employed to directly monitor the pro-angiogenic impact of the sponge material. Implantation of gelatin sponges onto the chorioallantoic membrane of fertilized chicken eggs induced robust attraction of endothelial cells and formation of blood vessels. Angiogenesis inside gelatin implants occurred more than 200 times faster than in a commercial collagen sponge. Similarly, after subcutaneous implantation of tube-like sponges into mice, an increasing immigration of cells and subsequent formation of functional vasculature became evident. Immunocytochemistry revealed no fibronection accumulation and no scarring. In summary, our matrix based on cross-linked gelatin promises to be a valuable component of future implants, improving neuronal and non-neuronal regeneration by concomitant pro-angiogenic stimulation.

Synthetic nerve guide implants in humans: a comprehensive survey.

OBJECTIVE: Lesions of the peripheral nervous system result in the loss of sensory and motor function and may in addition be accompanied by severe neuropathic syndromes originating from aberrant axonal regrowth. The transplantation of autologous nerve grafts represents the current "gold standard" during reconstructive surgery, despite obvious side effects. Depending on the demands of the lesion site, various donor nerves may be used for grafting (e.g., the sural, saphenous), sacrificing native functions in their target areas. Recently, several synthetic nerve guide implants have been introduced and approved for clinical use to replace autologous transplants. This alternative therapy is based on pioneering studies with experimental nerve guides. METHODS: We present a comprehensive review of all published human studies involving synthetic nerve guides. RESULTS: Data from some 300 patients suggest that for short nerve defects of a few centimeters, resorbable implants provide promising results, whereas a number of late compression syndromes have been documented for nonresorbable implants. CONCLUSIONS: To treat longer defects, further implant development is needed, a goal that could be achieved, for example, by more closely imitating the intact nerve architecture and regulatory cell-cell interactions.

Neuro tissue engineering of glial nerve guides and the impact of different cell types.

The aim of neuro tissue engineering is to imitate biological features in order to enhance regeneration. Following lesions of peripheral nerves, Schwann cells (SCs) reorganize to form longitudinal bands of Büngner (boB) which function as guides for regrowing axons. In order to imitate boB in synthetic implants designed to bridge nerve lesions, we developed resorbable, semipermeable nerve guide conduits with microstructured internal polymer filaments. We utilized a novel microcell chip and identified three extracellular matrix components conducive for coating non-permissive polymer surfaces. In order to maximize SC alignment, seven different microtopographies were investigated via the silicon chip technology. Special longitudinal microgrooves directed SC orientation and growing axons of dorsal root ganglia (DRG), thus achieving stereotropism. When these results were applied to microgrooved polymer filaments inside nerve guide conduits, we observed highly oriented axon growth without meandering. Since scar-forming fibroblasts could potentially interfere with axonal regrowth, triple cultures with fibroblasts, SC and DRG were conducted. Fibroblasts positioned on the outer nanopore containing conduit wall, did not hamper neuronal and glial differentiation inside the tube. In summary, for more rapid regrowth, functional boB can be induced by guided microtissue engineering. By considering both the negative and positive effects of cell interactions, a more rational design of nerve implants becomes feasible.