Spatially controlled delivery of neurotrophic factors in silk fibroin-based nerve conduits for peripheral nerve repair.

Restoration with sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. Silk has shown clinical promise for numerous tissue engineering applications due to its biocompatibility, impressive mechanical properties, and Food and Drug Administration approval. The aim of this study was to evaluate the efficacy of silk fibroin--based nerve guides containing glial cell line-derived neurotrophic factor (GDNF) in a long-gap sized (15 mm) rat sciatic nerve defect model. Four groups of nerve conduits were prepared: (1) silk conduits with empty silk microspheres, (2) silk conduits with GDNF-loaded silk microspheres uniformly distributed in the conduit wall, (3) silk conduits with GDNF-loaded silk microspheres in a controlled manner with the highest GDNF concentration at the distal end, and (4) isograft. After 6 weeks, the nerve grafts were explanted, harvested, and fixed for histologic analysis. Nerve tissue stained with the S-100, and neuroendocrine marker PGP 9.5 antibodies demonstrated a significantly increased density of nerve tissue in the GDNF-treated groups compared with the empty microsphere (control) group (P < 0.05). GDNF-treated animals with a higher concentration of GDNF in the distal portion possessed a significantly higher density of PGP 9.5 protein middle conduit part than comparison to GDNF uniform-treated animals (P < 0.05). Silk-based nerve conduits possess optimal mechanical and degradative properties, rendering them potentially useful in peripheral nerve repair. This study demonstrates that novel, porous silk fibroin--based nerve conduits, infused with GDNF in a controlled manner, represent a potentially viable conduit for Schwann cell migration and proliferation in the regeneration of peripheral nerves.

Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields.

BACKGROUND: Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood. METHODS: The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs) undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF) and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR) were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP) and collagen type 1 (col1), and stress response markers, such as heat shock protein 27 (hsp27) and heat shock protein 70 (hsp70). Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG) imaging. RESULTS: Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p < 0.05) in samples exposed to the electric field. A delayed but two fold increase in ALP and col1 transcript was detected by week 2 (p < 0.05) in differentiating hMSCs exposed to an electric field in comparison to the nonstimulated controls. Upregulation in stress marker, hsp27, and type 1 collagen deposition were correlated with this response. Increases in NADH, FAD, and lipofuscin were traced in the stimulation group during the first week of field exposure with differences statistically significant on day 10 (p < 0.05). Changes in hsp27 expression correlate well with changes in lipofuscin detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28. CONCLUSIONS: Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.