Methods. NVBs have been harvested and decellularized (SDS, Triton-X) and characterized (histology, immunohistochemistry, PicoGreen® DNA assay, collagenase assay, and differential scanning calorimetry). Samples were recellularized with human ECs and SCs, for 5 days in a bioreactor. Final endothelial integrity was verified by SEM and vascular casting.
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Conclusion. We have developed techniques for harvesting, decellularizing and recellularizing NVBs from xenogeneic sources. These novel "autologous" off-the-shelf NVBs aim to play an important role in reinnervation and revascularization for soft and hard tissue reconstruction, rehabilitation of massive scarring, and engineered tissues; and as free vascularized nerve grafts; while avoiding donor site morbidity and the need for immunosuppression.
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Introduction. Peripheral nerve injuries result in limited recovery, especially over large gaps. Nerve guidance conduits (NGCs) are an attractive option, but prove largely insufficient on their own. Creating a conducive micro-environment by incorporating bioactive moieties to aide speed and accuracy of regeneration, requires lengthy processes (screening, optimizing delivery, in vivo testing), which can take a year or longer. We describe a rapid "pump screening" model that delivers test compounds reliably over 4 weeks, without requiring any special formulation development. At 6 weeks, quantifiable histological outcomes are evaluated, dramatically reducing: study duration (6 vs. 16-24 weeks), in vivo cases (by 75%), and costs (by 68%).
Materials & Methods. Model: A 1 cm sciatic nerve gap is repaired with a tyrosine-derived polycarbonate NGC (1.5 mm ID x 12 mm) in a rat model. A drug-delivery cannula is affixed to the NGC, from a 2 ml Alzet® osmotic pump (DURECT, Cupertino, CA). The pump delivers drug throughout the NGC volume over every 20 hour period for 4 weeks. At 6 weeks histological quantification is used for analysis. Groups: There are 10 Experimental groups (test compounds that demonstrated promise in vitro, and remain efficacious for 4 weeks at 37 deg C); and 4 Control groups: (i, ii) Validation controls using hydrophilic (fluorescein, n=2) and hydrophobic (Nile red, n=2) compounds; (iii) Negative control (no drug, n=6); (iv) Contralateral control (normal nerve, n=6). Histology: Samples are stained with toluidine blue and osmium tetroxide. Cross-sections (XS) of proximal nerve (3 mm from NGC) and conduit (3 mm into proximal NGC), and longitudinal sections (LS) of the remaining NGC, are assessed for 6 quantifiable parameters: XS = axon count, density, diameter, g-ratio, nerve area; LS = propagation rate. Statistics: Experimental groups are currently each being compared to the negative control group, for each outcome (absolutely and for % change from proximal nerve into conduit). The Wilcoxon rank sum (Mann-Whitney) test is being used for each comparison since the sample sizes are small and the outcomes may not be normally distributed. For each outcome, a false discovery rate procedure is being used to address the multiple comparison situation. |
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